Multiplexed Analyses of Test Samples

ABSTRACT

The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer). The methods described create a nucleic acid surrogate for a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, especially protein targets. The disclosure further describes aptamer constructs that facilitate the use of aptamers in a variety of analytical detection applications.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/958,620, filed Dec. 2, 2010, entitled “Multiplexed Analyses of TestSamples”, which is a continuation-in-part application of U.S.application Ser. No. 12/175,446, filed Jul. 17, 2008, entitled“Multiplexed Analyses of Test Samples”. U.S. application Ser. No.12/175,446 claims the benefit of U.S. Provisional Application Ser. No.60/950,281, filed Jul. 17, 2007, U.S. Provisional Application Ser. No.60/950,293, filed Jul. 17, 2007, U.S. Provisional Application Ser. No.60/950,283, filed Jul. 17, 2007, U.S. Provisional Application Ser. No.61/031,420, filed Feb. 26, 2008 and U.S. Provisional Application Ser.No. 61/051,594, filed May 8, 2008. U.S. application Ser. No. 12/175,446is also a continuation in part of U.S. application Ser. No. 11/623,580and U.S. application Ser. No. 11/623,535, each of which was filed onJan. 16, 2007. Each of these applications is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods, devices, reagents,and kits for the detection of a target molecule in a sample and, morespecifically, to the detection and/or quantification of one or moretarget molecules that may be contained in a test sample. Such methodshave a wide utility in diagnostic applications as well as in biomarkerdiscovery and the design and development of therapeutics.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequence_Listing_ST25.txt”, created Nov. 30, 2010, size of onekilobyte.

BACKGROUND

The following description provides a summary of information relevant tothe present disclosure and is not a concession that any of theinformation provided or publications referenced herein is prior art tothe presently claimed invention.

Assays directed to the detection and quantification of physiologicallysignificant molecules in biological samples and other samples areimportant tools in scientific research and in the health care field. Oneclass of such assays involves the use of a microarray that includes oneor more aptamers immobilized on a solid support. The aptamers are eachcapable of binding to a target molecule in a highly specific manner andwith very high affinity. See, e.g., U.S. Pat. No. 5,475,096 entitled“Nucleic Acid Ligands” see also, e.g., U.S. Pat. No. 6,242,246, U.S.Pat. No. 6,458,543, and U.S. Pat. No. 6,503,715, each of which isentitled “Nucleic Acid Ligand Diagnostic Biochip”. Once the microarrayis contacted with a sample, the aptamers bind to their respective targetmolecules present in the sample and thereby enable a determination ofthe absence, presence, amount, and/or concentration of the targetmolecules in the sample.

A variation of this assay employs aptamers that include photoreactivefunctional groups that enable the aptamers to covalently bind or“photocrosslink” their target molecules. See, e.g., U.S. Pat. No.6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip”. Thesephotoreactive aptamers are also referred to as photoaptamers. See, e.g.,U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, and U.S. Pat. No.6,291,184, each of which is entitled “Systematic Evolution of NucleicAcid Ligands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539,entitled “Photoselection of Nucleic Acid Ligands”. After the microarrayis contacted with the sample and the photoaptamers have had anopportunity to bind to their target molecules, the photoaptamers arephotoactivated, and the solid support is washed to remove anynon-specifically bound molecules. Harsh wash conditions may be used,since target molecules that are bound to the photoaptamers are generallynot removed, due to the covalent bonds created by the photoactivatedfunctional group(s) on the photoaptamers. In this manner, the assayenables a determination of the absence, presence, amount, and/orconcentration of the target molecules in the test sample.

In both of these assay formats, the aptamers are immobilized on thesolid support prior to being contacted with the sample. Under certaincircumstances, however, immobilization of the aptamers prior to contactwith the sample may not provide an optimal assay. For example,pre-immobilization of the aptamers may result in inefficient mixing ofthe aptamers with the target molecules on the surface of the solidsupport, perhaps leading to lengthy reaction times and, therefore,extended incubation periods to permit efficient binding of the aptamersto their target molecules. Further, when photoaptamers are employed inthe assay and depending upon the material utilized as a solid support,the solid support may tend to scatter or absorb the light used to effectthe formation of covalent bonds between the photoaptamers and theirtarget molecules. Moreover, depending upon the method employed,detection of target molecules bound to their aptamers can be subject toimprecision, since the surface of the solid support may also be exposedto and affected by any labeling agents that are used. Finally,immobilization of the aptamers on the solid support generally involvesan aptamer-preparation step (i.e., the immobilization) prior to exposureof the aptamers to the sample, and this preparation step may affect theactivity or functionality of the aptamers.

Accordingly, a need exists for methods, devices, reagents, and kits thatprovide high sensitivity assays for the detection and/or quantificationof target molecules in a test sample by optimizing conditions thataffect one or more of the following: (1) the activity of aptamers, (2)the efficiency of achieving binding equilibria for aptamer-targetmolecule complexes, (3) the formation of covalent bond(s) between anaptamer and its target molecule, (4) removal of extraneous samplecomponents and excess aptamers, (5) dissociation of the affinity complexformed through the use of slow off-rate aptamers, and (6) the detectionof aptamer-target molecule complexes.

SUMMARY

The present disclosure includes methods, devices, reagents, and kits forthe detection and/or quantification of one or more target molecules thatmay be present in a test sample. More specifically, the disclosureprovides methods for the purification of aptamer affinity complexes (oraptamer covalent complexes), by removing both free target and freeaptamers from the aptamer affinity complexes (or aptamer covalentcomplexes), thereby removing potential sources of noise in the assay.The present disclosure also provides aptamer- and photoaptamer-basedassays for the quantification of target molecule wherein the aptamer (orphotoaptamer) can be separated from the aptamer affinity complex (orphotoaptamer covalent complex) for final detection using any suitablenucleic acid detection method. The disclosure also describes aptamerconstructs that facilitate the separation of the assay components fromthe aptamer affinity complex (or photoaptamer covalent complex) andpermit isolation of the aptamer for detection and/or quantification. Thedisclosure also describes methods, devices, kits, and reagents thatoffer improved sensitivity and specificity by employing aptamers thathave slow off-rates from their targets and improved bindingefficiencies. The present disclosure also provides methods, devices,kits, and reagents for the multiplexed analysis of a test sample,wherein multiple targets in the test sample may be simultaneouslydetected and/or quantified. Ultimately these methods and reagents allowfor the conversion of a target concentration (for example a proteintarget concentration in a test sample) to a nucleic acid concentrationthat can be detected and quantified by any of a wide variety of nucleicacid detection and quantification methods. Further, once the targetconcentration has been effectively converted to a corresponding nucleicacid concentration, standard nucleic acid amplification and detectionsteps can then be employed to increase the signal. Methods according tothe present disclosure may be conducted in vitro.

Single Catch Affinity Assay.

In one embodiment, a test sample is contacted with an aptamer that has aspecific affinity for a target molecule. If the test sample contains thetarget molecule, an aptamer affinity complex will form in the mixturewith the test sample. In one embodiment, a tag is attached to the targetmolecule of the aptamer affinity complex. (Note that the tag is designedsuch that it can be attached to the target in a manner that does notdisrupt the aptamer affinity complex.) In another embodiment, the tag isattached to the target prior to the formation of the aptamer affinitycomplex. In another embodiment, the tag is added to the target at anypoint prior to exposing the mixture to the capture element on the solidsupport. The tagged aptamer affinity complex is next captured on a solidsupport by exposing the mixture to the solid support. The attachment isaccomplished by contacting the solid support with the aptamer affinitycomplex and allowing the tag to associate either, directly orindirectly, with an appropriate capture agent that is attached to thesolid support. The aptamer affinity complex that has associated with thecapture agent on the solid support is partitioned from the remainder ofthe test sample mixture, thereby removing any free aptamer. The aptamersthat are complexed with the target in the aptamer affinity complex canbe released from the solid support by dissociation of the aptameraffinity complex. Finally, the released aptamers can be detected and/orquantified using any of a variety of suitable nucleic acid detectionmethods, including but not limited to mass spectrometry, the Invaderassay method, a nucleic acid chip, quantitative polymerase chainreaction (Q-PCR), and the like. In some embodiments, depending upon theparticular nucleic acid detection methods used, the aptamers may bedetected while still a part of the aptamer affinity complex.

Dual Catch Affinity Assay.

In another embodiment, a test sample is contacted with an aptamer thatincludes a releasable first tag and has a specific affinity for a targetmolecule. If the test sample contains the target molecule, an aptameraffinity complex will form in the mixture with the test sample. Theaptamer affinity complex is captured on a first solid support byexposing the mixture to the first solid support. The attachment isaccomplished by contacting a first solid support with the aptameraffinity complex and allowing the releasable first tag included on theaptamer to associate, either directly or indirectly, with an appropriatefirst capture agent that is attached to the first solid support. Notethat in addition to aptamer affinity complexes, uncomplexed aptamer willalso attach to the first solid support. The aptamer affinity complex anduncomplexed aptamer that has associated with the probe on the solidsupport is then partitioned from the remainder of the mixture, therebyremoving free target and all other uncomplexed matter in the test sample(sample matrix); i.e., components of the mixture not associated with thefirst solid support. Following partitioning the aptamer affinitycomplex, along with any uncomplexed aptamer, is released from the firstsolid support using a method appropriate to the particular releasablefirst tag being employed. A second tag (which may be the same ordifferent from the releasable first tag) is attached to the targetmolecule of the aptamer affinity complex. The second tag is designedsuch that it can be attached to the target in a manner that does notdisrupt the aptamer affinity complex. The aptamer affinity complex iscaptured on a second solid support by allowing the second tag toassociate either, directly or indirectly, with an appropriate secondcapture agent that is attached to a second solid support by exposing thereleased aptamer affinity complex to the second solid support. Theaptamer affinity complex that has associated with the probe on the solidsupport is partitioned from the remainder of the mixture, therebyremoving any free, uncomplexed, aptamer. The aptamers that are complexedwith the target in the aptamer affinity complex can be released from thesolid support by dissociation of the aptamer affinity complex. Finally,the aptamers that have been released from the aptamer affinity complexcan be detected and/or quantified using any of a variety of suitablenucleic acid methods, including but not limited to mass spectrometry,the Invader assay method, a DNA chip, quantitative polymerase chainreaction (Q-PCR), and the like. In some embodiments, the target may bereacted with the second tag while the aptamer affinity complex is stillimmobilized to the first solid support. Adding the second tag after thepartitioning step eliminates the labeling of target molecules that arenot part of an aptamer affinity complex. In some embodiments, where anucleic acid detection method is used, the aptamers may be detectedwhile still a part of the aptamer affinity complex.

Single Catch Photocrosslink Assay.

In another embodiment, a test sample is contacted with a photoaptamerthat has a specific affinity for a target molecule. If the test samplecontains the target molecule, a photoaptamer affinity complex will formin the mixture with the test sample. The aptamer affinity complex isconverted to an aptamer covalent complex by the appropriate excitationof the photocrosslinking group. A tag is attached to the target moleculeof the aptamer covalent complex. The tag is designed such that it can beattached to the target in a manner that does not disrupt the aptamercovalent complex. The aptamer covalent complex is captured on a solidsupport by exposing the mixture to the solid support. The attachment isaccomplished by contacting the solid support with the aptamer covalentcomplex and allowing the tag to associate either, directly orindirectly, with an appropriate capture agent that is attached to thesolid support. The aptamer covalent complex that has associated with thecapture agent on the solid support is partitioned from the remainder ofthe test sample mixture, thereby removing any free photoaptamer. Thephotoaptamer that is part of the aptamer covalent complex can bedetected and/or quantified (while still attached to the solid support)using any of a variety of methods, including but not limited to theInvader assay method, mass spectroscopy, a DNA chip, quantitativepolymerase chain reaction (Q-PCR), and the like.

In another embodiment, the Single Catch Photocrosslink Assay describedabove is modified such that, prior to detection of the photoaptamer, anucleic acid amplification step, such as, for example, polymerase chainreaction, is used to create one or more copies of the photoaptamers thatare a part of the aptamer covalent complexes that are bound to the solidsupport. These copies of the photoaptamers can then be released andsubsequently detected and/or quantified using any of a variety ofsuitable methods, including but not limited to mass spectrometry, theInvader assay method, a DNA chip, quantitative polymerase chain reaction(Q-PCR), and the like.

In another embodiment of the Single Catch Photocrosslink Assay, thephotocrosslinking group of the photoaptamer is attached to the aptamervia a cleavable linker. In one embodiment, this cleavable linker is aphotocleavable linker, but may be a chemically cleavable linker or anyother cleavable linker that can be cleaved to release the targetmolecule from the tag at any desirable point in the assay. In thisembodiment, the Single Catch Photocrosslink Assay described above ismodified such that, prior to detection of the photoaptamer, thecleavable linker is used to release the photoaptamer from thephotoaptamer covalent complex that is bound to the solid support. Thereleased aptamers can be detected and/or quantified using any of avariety of suitable methods, including but not limited to massspectrometry, the Invader assay method, a DNA chip, quantitativepolymerase chain reaction (Q-PCR), and the like.

In yet another embodiment of the Single Catch Photocrosslink Assay, thetag that is attached to the target molecule is attached via a cleavablelinker. In one embodiment, this cleavable linker is a photocleavablelinker. In other embodiments of this assay, the tag is attached via achemically cleavable linker or any other suitable cleavable linker thatcan be cleaved to release the target molecule from the tag at anydesirable point in the assay. In this embodiment, the Single CatchPhotocrosslink Assay described above is modified such that, prior todetection of the photoaptamer, the cleavable linker is used to releasethe aptamer covalent complex from the solid support. The releasedaptamer covalent complex can be detected and/or quantified using any ofa variety of suitable methods, including but not limited to massspectrometry, the Invader assay method, a DNA chip, quantitativepolymerase chain reaction (Q-PCR), and the like.

Dual Catch Photocrosslink Assay.

In another embodiment, a test sample is contacted with a photoaptamerthat contains a first releasable tag and that has a specific affinityfor a target molecule. If the test sample contains the target molecule,a photoaptamer affinity complex will form in the mixture with the testsample. The photoaptamer affinity complex is converted to an aptamercovalent complex by the appropriate excitation of the photocrosslinkinggroup. The aptamer covalent complex is captured on a first solid supportby exposing the mixture to the first solid support. The attachment isaccomplished by contacting a first solid support with the aptamercovalent complex and allowing the releasable first tag included on thephotoaptamer to associate either, directly or indirectly, with anappropriate first capture agent attached to the first solid support.Note that in addition to photoaptamer covalent complexes, uncomplexedphotoaptamers may also attach to the solid support. The aptamer covalentcomplex and uncomplexed aptamer that has associated with the probe onthe solid support is partitioned from the remainder of the mixture,thereby removing free target and all other uncomplexed matter in thetest sample (sample matrix). Following partitioning the photoaptamercovalent complex, along with any uncomplexed photoaptamer, is releasedfrom the solid support using a method appropriate to the particularreleasable first tag being employed. A second tag is attached to thetarget molecule of the aptamer covalent complex. The second tag isdesigned such that it can be attached to the target in a manner thatdoes not disrupt the aptamer covalent complex. The aptamer covalentcomplex is captured on a second solid support by exposing the releasedaptamer covalent complex to the second solid support. The attachment isaccomplished by contacting the second solid support with the aptamercovalent complex and allowing the second tag to associate either,directly or indirectly, with an appropriate second capture agentattached to the second solid support. The aptamer covalent complex thathas associated with the second capture agent on the solid support ispartitioned from the remainder of the mixture, thereby removing any freephotoaptamer. The photoaptamer that is part of the aptamer covalentcomplex can be detected and/or quantified (while still attached to thesolid support) using any of a variety of suitable methods, including butnot limited to the Invader assay method, mass spectroscopy, a DNA chip,quantitative polymerase chain reaction (Q-PCR), and the like.

In another embodiment, the Dual Catch Photocrosslink Assay describedabove is modified such that, prior to detection, a nucleic acidamplification step such as, for example, polymerase chain reaction, isused to create one or more copies of the photoaptamers that are a partof the aptamer covalent complex that is bound to the solid support.These copies of the photoaptamer can be released and subsequentlydetected and/or quantified using any of a variety of suitable methods,including but not limited to mass spectrometry, the Invader assaymethod, a DNA chip, quantitative polymerase chain reaction (Q-PCR), andthe like.

In another embodiment, the Dual Catch Photocrosslink Assay describedabove is modified such that the photocrosslinking group of thephotoaptamer is attached to the aptamer via a cleavable linker. In oneembodiment, this cleavable linker is a photocleavable linker. In otherembodiments of this assay, the photocrosslinking group of thephotoaptamer is attached to the aptamer via a chemically cleavablelinker or any other suitable cleavable linker that can be cleaved torelease the photocrosslinking group from the photoaptamer covalentcomplex at any desirable point in the assay. In this embodiment, theDual Catch Photocrosslink Assay described above is modified such that,prior to detection of the photoaptamer, the cleavable linker is used torelease the photoaptamer from the photoaptamer covalent complex that isbound to the solid support. The released photoaptamers can be detectedand/or quantified using any of a variety of suitable methods, includingbut not limited to mass spectrometry, the Invader assay method, a DNAchip, quantitative polymerase chain reaction (Q-PCR), and the like.

In yet another embodiment, in the Dual Catch Photocrosslink Assay thetag that is attached to the target molecule is attached via a cleavablelinker. In one embodiment, this cleavable linker is a photocleavablelinker. In other embodiments of this assay, the tag is attached via achemically cleavable linker or any other suitable cleavable linker thatcan be cleaved to release the target molecule from the tag at anydesirable point in the assay. In this embodiment, the Dual CatchPhotocrosslink Assay described above is modified such that, prior todetection of the photoaptamer, the cleavable linker is used to releasethe photoaptamer covalent complex from the solid support. The releasedphotoaptamer covalent complex can be detected and/or quantified usingany of a variety of suitable methods, including but not limited to massspectrometry, the Invader assay method, a DNA chip, quantitativepolymerase chain reaction (Q-PCR), and the like.

Kinetic Challenge.

In another embodiment, a kinetic challenge may be used to increase thespecificity and sensitivity of the assays disclosed herein. The kineticchallenge, first described in U.S. application Ser. No. 11/623,580 andU.S. application Ser. No. 11/623,535, each of which was filed on Jan.16, 2007 and the contents of both of which are incorporated by referenceherein in their entireties, provides for using the relatively longoff-rates of specific aptamer target complexes relative to non-specificcomplexes to increase the specificity of certain assays. Furthermore,U.S. application Ser. No. 12/175,434, filed Jul. 17, 2008 entitled“Method for Generating Aptamers with Improved Off-Rates”, which isincorporated herein by reference in its entirety, discloses that slowoff-rate aptamers can be identified by employing a slow off-rateenrichment process during the SELEX process and/or by using certainmodified nucleotides. (See, U.S. application Ser. No. 12/175,388, filedJul. 17, 2008 entitled “Improved SELEX and PhotoSELEX” which isincorporated herein by reference in its entirety).

The above described assays (the Single Catch Affinity Assays, the DualCatch Affinity Assays, the Single Catch Photocrosslink Assay, and theDual Catch Photocrosslink Assay) can each be improved through theincorporation of a kinetic challenge. For purposes of illustration only,the following describes how a kinetic challenge can be added to selectedembodiments of the Dual Catch Affinity Assay and the Dual CatchPhotocrosslink Assay. It should be understood that a kinetic challengecan be added to the any of other assays and methods described herein ina similar manner. It should be further understood that the kineticchallenge can be added at any suitable point in any of the describedassays and methods in addition to the places (steps) noted in thevarious embodiments described herein.

In one embodiment, a kinetic challenge is inserted into the Dual CaptureAffinity Assay after the step in which the aptamer affinity complex anduncomplexed aptamer that has associated with the probe on the firstsolid support is partitioned from the remainder of the mixture andbefore the step in which the aptamer in the aptamer affinity complex iseither released or is directly detected or quantified. In oneembodiment, the kinetic challenge is performed after the aptameraffinity complex is released from the first solid support. In thisembodiment, the kinetic challenge is performed by releasing the aptameraffinity complex into a buffer that contains a high concentration of acompetitor and subsequently incubating the aptamer affinity complexes inthe competitor solution for a time less than or equal to thedissociation half life of the aptamer affinity complex.

In another embodiment, a kinetic challenge is inserted into the DualCapture Photocrosslink Assay after the formation of the aptamer affinitycomplex and before the crosslinking step. In one embodiment, the kineticchallenge is performed by adding a competitor to the mixture containingthe aptamer affinity complex and subsequently incubating the aptameraffinity complexes in the competitor solution for a time less than orequal to the dissociation half life of the aptamer affinity complex.

Detection and Quantification Methods.

As mentioned above, it is possible to detect the aptamer affinitycomplex (or aptamer covalent complex in the case of the photocrosslinkassays) by employing a number of different nucleic acid detectiontechniques, including mass spectrometry, the Invader assay, DNA chips,quantitative PCR methods, and the like.

In one embodiment, the aptamer affinity complex (or aptamer covalentcomplex) is detected and/or quantified using a DNA chip. In thisembodiment, the aptamer affinity complex (or aptamer covalent complex)that has associated with the solid support is eluted and is hybridizedto a complementary probe sequence that has been printed on a DNA chip.In one embodiment, the complementary probe sequence is complementary tothe entire aptamer. In another embodiment, the complementary probesequence is complementary to only a portion of the aptamer. In anotherembodiment, the probe is complementary to a sequence added to theaptamer for the purpose of hybridization. In order to detect thehybridized aptamer (or photoaptamer) on the DNA chip, a label can beintroduced. In one embodiment, the label is incorporated into theaptamer at the time the aptamer (or photoaptamer) is synthesized. Forexample, a fluorescent dye can be incorporated into a chemically (orenzymatically) synthesized aptamer. In one embodiment, a label is addedto the aptamer during synthesis of the aptamer. In other embodiments, alabel is added to the aptamer at any time before, during or after theassay. In another embodiment, nucleic acid amplification techniques suchas PCR can be used to amplify the aptamer (or photoaptamer) population.In this case, a label can also be incorporated as a part of theamplification step.

In another embodiment, the aptamer affinity complex (or aptamer covalentcomplex) is detected and/or quantified using mass spectrometry. In thisembodiment, the aptamer affinity complex (or aptamer covalent complex)that has associated with the solid support is eluted and analyzed usingmass spectrometry, which produces a spectrum of peaks that can be usedto identify, and therefore detect, the target molecule. Once the targetmolecule has been detected, optionally it can also be quantified by anynumber of suitable techniques. In one embodiment, where the targetmolecule is a protein or polypeptide, prior to using mass spectrometryto analyze the aptamer affinity complex (or aptamer covalent complex),the aptamer affinity complex (or aptamer covalent complex) can bedigested with protease enzymes, such as, for example, proteinase K ortrypsin, to produce fragments of the bound target molecule that can beused to identify the target molecule, and thereby enable detection andoptional quantification of the target molecule.

In another embodiment, the aptamer affinity complex (or aptamer covalentcomplex) is detected and/or quantified using Q-PCR. As mentioned above,this can be done either while the aptamer affinity complex (or aptamercovalent complex) is attached to the solid support or after release fromthe solid support. The aptamer affinity complex (or aptamer covalentcomplex) is quantified by performing PCR and determining, eitherdirectly or indirectly, the amount or concentration of aptamer that hadbound to its target molecule in the test sample. The amount orconcentration of the target molecule in the test sample is generallydirectly proportional to the amount or concentration of the aptamerquantified by using Q-PCR. An exemplary method that may be employed toquantify an aptamer affinity complex (or aptamer covalent complex) inthis manner is the TaqMan® assay (PE Biosystems, Foster City, Calif.;see also U.S. Pat. No. 5,210,015).

In another embodiment, the aptamer is optionally dissociated from itscorresponding target molecule, prior to detection and/or quantification.The free aptamer can be detected and measured using any known suitablemethod for the detection and/or quantification of nucleic acids.

Multiplexed Assays.

In another embodiment, the assays and methods described above are usedto detect and/or quantify two or more targets. In one embodiment,multiple aptamers are used in the Dual Capture Affinity Assay toquantify and/or detect multiple targets. After final release of theaptamers from the aptamer affinity complexes, each of the aptamers canthen be detected using suitable methods for the multiplexed detection ofnucleic acids. In one method, a multiplexed DNA chip is used to detectand/or quantify the aptamers. Any of the assays disclosed herein can beperformed in a multiplexed fashion to detect multiple targets. Becausethere are no inherent limits to the scale of the multiplexing, thesemultiplexed assays can be used to detect, for example, 2 or moretargets, 10 or more targets, 25 or more targets, 50 or more targets, 100or more targets, 250 or more targets, 500 or more targets, or 1000 ormore targets.

Reagents and Kits.

In one embodiment, kits for various detection applications, includingwithout limitation diagnostic kits, biomarker discovery kits,environmental testing kits, biohazard or bioweapons detection kits andkits for detecting targets in life science and analytical chemistryapplications, can be prepared based on the methods disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate exemplary methods for the detection and/orquantification of one or more target molecules that may be present in atest sample.

FIGS. 2A and 2B illustrate exemplary methods for the detection and/orquantification of one or more target molecules that may be present in atest sample.

FIGS. 3A-3L illustrate exemplary aptamer constructs for use with theassays described herein.

FIG. 4 presents a list of over 500 targets for which aptamers have beenproduced. Many of these aptamers have been designed to have slowdissociation rates from their respective target.

FIG. 5A illustrates an example of a hybridization tag. FIGS. 5B to 5Dillustrate examples of aptamer constructs including a cleavable orreleasable element, a tag (for example biotin), a spacer, and a label(for example Cy3).

FIG. 6 illustrates the aptamer and primer constructs used in the assaymethods described in this disclosure. Cy3 represents a Cyanine 3 dye, Ba biotin, PC a photocleavable linker, ANA a photoreactive crosslinkinggroup, (AB)₂ a pair of biotin residues separated by dA residues, and(T)₈ a poly dT linker. Primer constructs are complementary to thecomplete 3′ fixed region of the aptamer constructs.

FIG. 6A. Aptamer construct used in the Single Catch Affinity AssayProtocol.

FIG. 6B. Aptamer construct used in the Dual Catch Affinity AssayProtocol.

FIG. 6C. Aptamer construct used in the Single Catch Crosslinking AssayProtocol.

FIG. 6D. Aptamer construct used in the Dual Catch Crosslinking AssayProtocol.

FIGS. 7A, 7B and 7C illustrate dose response curves (RFU vs log inputtarget protein concentration) for detection of target proteins in bufferusing the Affinity Assay Protocol with Microarray Detection. Replicateno-protein control values are plotted on the y-axis frame. The solidlines represent a sigmoidal fit through the data points. The dashedlines represent two standard deviations of the replicate no-proteinvalues. FIG. 7A. bFGF target protein. FIG. 7B. FGF7 target protein. FIG.7C. Lymphotactin target protein.

FIG. 8 illustrates the dose response curves for three replicatemeasurements of the target protein Lymphotactin in buffer using theAffinity Assay Protocol with Microarray Detection. Replicate no-proteincontrol values are plotted on the y-axis frame. The solid linesrepresent sigmoidal fits through the data points for each of the threereplicates.

FIG. 9 illustrates the dose response curve (RFU versus log input targetprotein concentration) for detection of the target protein Lymphotactinin 10% human plasma using the Affinity Assay Protocol and MicroarrayDetection. Replicate no-protein control values are plotted on the y-axisframe and circled. The solid line represents a sigmoidal fit through thedata points.

FIG. 10 illustrates the dose response curve (RFU versus log input targetprotein concentration) for detection of the target protein Lymphotactinin 10% whole human blood using the Affinity Assay Protocol andMicroarray Detection. Replicate no-protein control values are plotted onthe y-axis frame and circled. The solid line represents a sigmoidal fitthrough the data points.

FIG. 11 illustrates the dose response curve (RFU versus log input targetprotein concentration) for the detection of the target proteinAngiogenin in buffer using the Photo-Crosslink Assay Protocol withMicroArray Detection. The solid line represents a sigmoidal fit throughthe data points. Four replicate no-protein data points are circled.

FIG. 12 illustrates the dose response curve (detected aptamerconcentration versus input target protein concentration) for detectionof Angiogenin in buffer using the Affinity Assay Protocol with Q-PCRDetection. Four replicate no-protein measurements are indicated on they-axis as open circles.

FIGS. 13A to 13C illustrate dose response curves for slow off-rateaptamers versus traditional aptamers for three different targets.

FIG. 14 illustrates the sample layout for an assay reproducibilitystudy.

FIG. 15 illustrates the CV's of the pooled and unpooled sample study.

FIG. 16 exhibits the base modifications of nucleotides discussed in thisdisclosure. The R groups that may be used are described in addition tothe linkers (X) that may be used between the nucleotide attachment pointand the R group is shown. The positions of attachment for the various“R” groups are also indicated on the respective R groups.

DETAILED DESCRIPTION

The practice of the current invention employs, unless otherwiseindicated, conventional methods of chemistry, microbiology, molecularbiology, and recombinant DNA techniques within the level of skill in theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition).

All publications, published patent documents, and patent applicationscited in this specification are indicative of the level of skill in theart(s) to which the invention pertains. All publications, publishedpatent documents, and patent applications cited herein are herebyincorporated by reference to the same extent as though each individualpublication, published patent document, or patent application wasspecifically and individually indicated as being incorporated byreference.

The present disclosure includes improved methods, devices, reagents, andkits for the detection and/or quantification of one or more targetmolecules that may be present in a test sample. The disclosed methods,devices, reagents, and kits provide high sensitivity assays for thedetection and/or quantification of target molecules in a test sample byoptimizing conditions that affect one or more of (1) the activity ofaptamers, (2) the efficiency of achieving binding equilibria foraptamer-target molecule complexes, (3) the formation of covalent bond(s)between an aptamer and its target molecule, (4) the removal of excessreagents and sample components, (5) the use of slow off rate aptamers,(6) desirable aptamer constructions, and (7) the detection ofaptamer-target molecule complexes.

It is noteworthy that, unless otherwise specified in a particularembodiment, the methods for the detection and/or quantification of atarget molecule described herein are independent of the specific orderin which the steps are described. For purposes of illustration, themethods are described as a specific sequence of steps; however, it is tobe understood that any number of permutations of the specified sequenceof steps is possible, so long as the objective of the particular assaybeing described is accomplished. Stated another way, the steps recitedin any of the disclosed methods may be performed in any feasible order,and the methods of the invention are not limited to any particular orderpresented in any of the described embodiments, the examples, or theappended claims. Further, for convenience and ease of presentation, thevarious methods are described with reference to a single target moleculeand a single aptamer. However, it is to be understood that any of thedescribed methods can be performed in a multiplex format that canprovide for the simultaneous detection and/or quantification of multipletargets using multiple aptamers, such that, for example, multiple targetmolecules in a test sample can be detected and/or quantified bycontacting the test sample with multiple aptamers, wherein each aptamerhas a specific affinity for a particular target molecule (i.e., in amultiplex format).

With reference to FIGS. 1A and 1B, the presence of a target molecule ina test sample is detected and/or quantified by first contacting a testsample with an aptamer that has a specific affinity for a targetmolecule. The method may be applied to the detection and/orquantification of a number of specific targets by use of thecorresponding number of specific aptamers, i.e. a multiplex format. Asingle target discussion is presented only for ease of presentation. Anaptamer affinity complex is formed by the aptamer binding to its targetmolecule if the test sample contains the target molecule. The aptameraffinity complex is optionally converted, using a method appropriate tothe aptamer being employed, to an aptamer covalent complex where theaptamer is covalently bound to its target molecule. A partition step isthen employed to remove free aptamer. The aptamer affinity complex (oraptamer covalent complex) is detected and/or quantified. A number ofdifferent detection methods can be used to detect the aptamer affinitycomplex, for example, the Invader assay, hybridization assays or DNAchips, mass spectroscopy, or Q-PCR.

As discussed above, the assays described here have been grouped for easein presentation into 4 assay formats: Single Catch Affinity Assays; DualCatch Affinity Assays, Single Catch Photocrosslink Assays; and DualCatch Photocrosslink Assays. However, it should be understood that othergroupings, combinations, and ordering of steps are contemplated and allfall within the scope of the disclosure. All four assay formats share acommon step in which aptamer-target complexes are separated from freeaptamer (or free photoaptamer) by a partitioning step that captures thetarget, such as, for example, protein. This partitioning step isreferred to herein as the “Catch 2” partition. The two “dual-catch”assays share an additional commonality in which aptamer-target complexesare separated from free target by a partitioning step that captures theaptamer. This latter partitioning step is referred to herein as the“Catch 1” partition. Methods for implementing each of these steps aredescribed in detail below.

The use of a kinetic challenge in each of these assay formats is furtherdisclosed. Traditionally, specificity in the detection of a desiredtarget has been improved through the use of a sandwich assay in whichtwo capture reagents are used. It has surprisingly been observed thatthe application of a kinetic challenge to a detection procedureemploying aptamers eliminates the need to enhance specificity byintroducing a second capture reagent. If a kinetic challenge isintroduced, non-specific complexes between the aptamer and anynon-target molecules are unlikely to re-form following theirdissociation. Since non-specific complexes generally dissociate morerapidly than an aptamer affinity complex, a kinetic challenge reducesthe likelihood that an aptamer will be involved in a non-specificcomplex with a non-target. An effective kinetic challenge can providethe assay with additional specificity, beyond that of the initialaptamer binding event and any subsequent covalent interaction. Thus, thekinetic challenge offers a second determinant of specificity in thesedetection methods. Methods for implementing the kinetic challenge aredescribed in detail below.

With reference to FIGS. 2A (dual step affinity assay) and 2B (dual stepcrosslinking assay), in an exemplary method for the detection and/orquantification of a target molecule that may be present in a testsample, a test sample is contacted with an aptamer (or photoaptamer)that includes a first tag and has a specific affinity for a targetmolecule. An aptamer affinity complex that includes an aptamer (orphotoaptamer) bound to its target molecule is allowed to form, where thetest sample contains the target molecule. In the photocrosslinkingexample (2B), the aptamer affinity complex is converted, using a methodappropriate to the aptamer being employed, to an aptamer covalentcomplex where the photoaptamer is covalently bound to its targetmolecule. The aptamer affinity complex (or aptamer covalent complex) isattached to a first solid support via a first capture element. Theattachment is accomplished by contacting the first solid support withthe aptamer affinity complex (or aptamer covalent complex) and allowingthe tag included on the aptamer to associate either, directly orindirectly, with a first capture element that is attached to the firstsolid support. The aptamer affinity complex (or aptamer covalentcomplex) that has associated with the first capture element on the firstsolid support is partitioned from the remainder of the mixture.Following partitioning the aptamer affinity complex (or aptamer covalentcomplex) is released from the first solid support using a methodappropriate to the particular tag being employed. Alternatively, the tagcan be attached the aptamer via a cleavable moiety, where such cleavablemoiety is now cleaved to release the aptamer affinity (or aptamercovalent complex) from the first solid support. A second tag (which maybe the same or different from the first tag) is attached to the targetmolecule of the aptamer affinity complex (or aptamer covalent complex).Optionally, a kinetic challenge can be performed to increase the assayspecificity and decrease the background signal. The aptamer affinitycomplex (or aptamer covalent complex) is attached to a second solidsupport. The attachment is accomplished by contacting the second solidsupport with the aptamer affinity complex (or aptamer covalent complex)and allowing the second tag included on the target to associate either,directly or indirectly, with a second capture element that is attachedto the second solid support. The aptamer affinity complex (or aptamercovalent complex) that has associated with the second capture element onthe second solid support is partitioned from the remainder of themixture. The aptamer affinity complex (or aptamer covalent complex) isdetected and optionally quantified.

In another embodiment, the aptamer (or photoaptamer) is firstdissociated from its respective target molecule and the free aptamer (orphotoaptamer) is detected and optionally quantified.

The aptamer affinity complex can be detected by utilizing any suitablenucleic acid detection technique, such as, for example, hybridization ora DNA chip, Q-PCR, mass spectroscopy (MS), the Invader assay, and thelike. Depending on which technique is employed, the aptamer may bedesigned or modified to include a label. This can be accomplished duringsynthesis (either enzymatically or chemically) or at any time during theassay (i.e., at any time prior to detection).

The methods disclosed herein enable the detection of the presence andamount of a target molecule by detecting free aptamer eluted from theassay. This allows for convenient detection and quantification of thetarget molecule, due to the relative simplicity of detection,quantification, and amplification of nucleic acids, and provides targetdetection assays that have a very favorable signal-to-noise ratio.

Single Catch (Catch 2-Only) Affinity Assay

In one embodiment, a single catch affinity assay is performed using acatch 2 partition, see FIG. 1A or 1B. This method works well when thesample matrix is not very complex so that other components in the sampledo not compete for the tag. It also works well for samples in which thetarget is present in high copy number or concentration.

Aptamers having high affinity and specificity for a target molecule areprovided. In one embodiment, the aptamer construct illustrated in FIG.6A is used. In this embodiment, the aptamer is contacted with a samplethat may contain a target molecule to form a mixture containing theaptamer, the target molecule, and non-target molecules, or samplematrix. Where the target molecule is present in the sample, aptameraffinity complexes are formed. The mixture may optionally be incubatedfor a period of time sufficient to achieve equilibrium binding of theaptamer to the target molecule, (e.g., at least about 10 minutes, atleast about 20 minutes, at least about 30 minutes).

In one embodiment, the mixture may optionally be subject to a kineticchallenge. The kinetic challenge helps reduce any non-specific bindingbetween the aptamer and any non-target molecules present in the sample.In one embodiment, 10 mM dextran sulfate is added and the mixture isincubated for about 15 minutes.

In one embodiment, the catch-2 partition is performed to remove freeaptamer. In one embodiment, the mixture containing aptamer affinitycomplexes is treated with an agent that introduces a capture tag to thetarget molecule component of the aptamer affinity complexes. In otherembodiments, the tag is introduced before the aptamer is contacted withthe text mixture, either before equilibrium binding or before thekinetic challenge. In one embodiment, the target is a protein orpeptide, and a biotin tag is attached to the target molecule by treatingwith NHS-PEO4-biotin. The mixture is then contacted with a solidsupport, that has a capture element adhered to its surface which iscapable of binding to the target capture tag. In this embodiment, thecapture element on the solid support is typically selected such that itbinds to the target capture tag with high affinity and specificity. Inone embodiment, the solid support is magnetic beads (such as DynaBeadsMyOne Streptavidin C1) contained within a well of a microtiter plate andthe capture element is streptavidin. The magnetic beads provide aconvenient method for the separation of partitioned components of themixture. Aptamer affinity complexes contained in the mixture are therebybound to the solid support through the binding interaction of the targetcapture tag and capture element on the solid support. The aptameraffinity complex is then partitioned from the remainder of the mixture,e.g. by washing the support to remove non-complexed aptamers. In oneembodiment, aptamer from the aptamer affinity complex can then bereleased for further processing by one or more of the followingtreatments: high salt, high pH, low pH or elevated temperature.

In another embodiment, the aptamer released from the catch-2 partitionis detected and optionally quantified by any suitable nucleic aciddetection methods, such as, for example, DNA chip hybridization, Q-PCR,mass spectroscopy, the Invader assay, and the like. In anotherembodiment the aptamer in the aptamer affinity complex is detected andoptionally quantified while still in contact with the solid support. Inone embodiment, the aptamer comprises a detectable moiety to facilitatethis detection step. The detectable moiety is chosen based on thedetection method to be employed. In one embodiment, the detectablemoiety or label is added to the aptamer during synthesis or prior to theassay. In another embodiment, the detectable moiety is added to theaptamer either during the assay or during the detection. The detectedaptamer can then be correlated with the amount or concentration oftarget in the original test sample.

Dual Catch (Catch 1 & 2) Affinity Assay

In one embodiment, a dual catch affinity assay is similar to the singlecatch affinity assay, but with an additional partitioning step toprovide additional sensitivity and specificity. In one embodiment,aptamers having high affinity and specificity for a target molecule andhaving a first releasable tag are provided. In another embodiment, thefirst releasable tag is added at any time in the assay prior to thecatch 1 partition, see FIGS. 2A and 2B. In one embodiment, this firstreleasable tag is a photocleavable biotin. In one embodiment, theaptamer construct illustrated in FIG. 6B is used. These and other tagsand cleavable moieties and aptamer containing such tags and cleavablemoieties are described. The aptamer is contacted with a sample that maycontain a target molecule to form a mixture containing the aptamer, thetarget molecule, and non-target molecules, or sample matrix. Where thetarget molecule is present in the sample, aptamer-target moleculecomplexes (aptamer affinity complexes) are formed. The mixture mayoptionally be incubated for a period of time sufficient to achieveequilibrium binding of the aptamer to the target molecule (e.g., atleast about 10 minutes, at least about 20 minutes, at least about 30minutes).

In one embodiment, the catch 1 partition is performed to remove any freetarget and sample matrix. The mixture is contacted with a first solidsupport having a first capture element adhered to its surface which iscapable of binding to the aptamer capture tag, preferably with highaffinity and specificity. In one embodiment, the first releasable tag isa photocleavable biotin, the first solid support is agarose beads in acolumn and the capture element is streptavidin. For example, PierceImmobilized Streptavidin beads may be used. Aptamer affinity complexescontained in the mixture are thereby bound to the first solid supportthrough the binding interaction of the first releasable tag and firstcapture element. The aptamer affinity complexes are partitioned from theremainder of the mixture, e.g. by washing the first solid support toremove non-bound molecules.

In one embodiment, aptamer affinity complexes bound to the solid supportare then treated with an agent that introduces a second tag to thetarget molecule component of the aptamer affinity complexes. In oneembodiment, the target is a protein or a peptide, and the target isbiotinylated by treating it with NHS-PEO4-biotin. The second tagintroduced to the target molecule may be the same as or different fromthe aptamer capture tag. If the second tag is the same as the first tag,or the aptamer capture tag, free capture sites on the first solidsupport may be blocked prior to the initiation of this tagging step. Inthis exemplary embodiment, the first solid support is washed with freebiotin prior to the initiation of target tagging. Tagging methods, andin particular, tagging of targets such as peptides and proteins aredescribed. In other embodiments, tagging of the target is performed atany other point in the assay prior to initiation of the catch 2partitioning, see FIGS. 2A and 2B. When the first and second tags arethe same, the target is tagged after the capture step of the catch 1partitioning has been performed.

Catch 1 partitioning is completed by releasing of the aptamer affinitycomplexes from the first solid support. In one embodiment, the firstreleasable tag is a photocleavable moiety that is cleaved by irradiationwith a UV lamp under conditions that cleave ≧ about 90% of the firstreleasable tag. In other embodiments, the release is accomplished by themethod appropriate for the selected releasable moiety in the firstreleasable tag. Aptamer affinity complexes may be eluted and collectedfor further use in the assay or may be contacted with another solidsupport to conduct the remaining steps of the assay (FIGS. 2A and 2B).

In one embodiment, the mixture may optionally be subject to a kineticchallenge. The kinetic challenge helps reduce any non-specific bindingbetween aptamers and non-target molecules. In one embodiment, 10 mMdextran sulfate is added to the aptamer affinity complexes, and themixture is incubated for about 15 minutes. In another embodiment, thekinetic challenge is initiated by performing the catch 1 elution in thepresence of 10 mM dextran sulfate. In other embodiments, the kineticchallenge is performed after the equilibrium binding step and before thecatch 2 partitioning.

In one embodiment, the catch-2 partition is performed to remove freeaptamer. As described above, in one embodiment, a second tag used in thecatch-2 partition may be added to the target while the aptamer affinitycomplex is still in contact with the solid support used in the catch-1partition. In other embodiments, the second tag may be added to thetarget at another point in the assay prior to initiation of catch 2partitioning. The mixture is contacted with a solid support, the solidsupport having a capture element (second) adhered to its surface whichis capable of binding to the target capture tag (second tag), preferablywith high affinity and specificity. In one embodiment, the solid supportis magnetic beads (such as DynaBeads MyOne Streptavidin C1) containedwithin a well of a microtiter plate and the capture element (secondcapture element) is streptavidin. The magnetic beads provide aconvenient method for the separation of partitioned components of themixture. Aptamer affinity complexes contained in the mixture are therebybound to the solid support through the binding interaction of the target(second) capture tag and the second capture element on the second solidsupport. The aptamer affinity complex is then partitioned from theremainder of the mixture, e.g. by washing the support to removenon-complexed aptamers. In one embodiment, aptamer from the aptameraffinity complex can then be released for further processing by one ormore of the following treatments: high salt, high pH, low pH or elevatedtemperature.

In another embodiment, the aptamer released from the catch-2 partitionis detected and optionally quantified by any suitable nucleic aciddetection methods, such as, for example, DNA chip hybridization, Q-PCR,mass spectroscopy, the Invader assay, and the like. These detectionmethods are described in further detail below. In another embodiment theaptamer in the aptamer affinity complex is detected and optionallyquantified while still in contact with the solid support. In oneembodiment, the aptamer comprises a detectable moiety to facilitate thisdetection step. The detectable moiety is chosen based on the detectionmethod to be employed. In one embodiment, the detectable moiety (label)is added to the aptamer during synthesis or any time prior to the assay.In another embodiment, the detectable moiety is added to the aptamereither during the assay or during the detection. The detected aptamercan be correlated to the amount or concentration of the target in thetest sample.

Single Catch (Catch 2-Only) Crosslinking Assay

In one embodiment, a single catch crosslinking assay is performed usinga catch 2 partition. This method works well when the sample matrix isnot very complex so that other components in the sample do not competefor the tag. It also works well for samples in which the target ispresent in high copy number or concentration. In some cases, additionalbenefit is provided by forming a covalent link between the photoaptamerand the target as it may allow more stringent washes in steps after thecrosslinking has been performed (FIG. 1B).

Photoaptamers having high affinity and specificity for a target moleculeare provided. In one embodiment, the crosslinking moiety of thephotoaptamer is linked to the aptamer via a cleavable linker. In oneembodiment, the crosslinking group is ANA (4-azido-2-nitro-aniline) andthe photocleavable group is PC Linker. In one embodiment, the aptamerconstruct illustrated in FIG. 6C is used. The photoaptamer is contactedwith a sample that may contain target molecules to form a mixturecontaining the aptamer, the target molecule, and non-target molecules,or sample matrix. Where the target molecule is present in the sample,(photo)aptamer affinity complexes are formed. The mixture may optionallybe incubated for a period of time sufficient to achieve equilibriumbinding of the aptamers and target molecules, (e.g., for at least about10 minutes, at least about 20 minutes, or at least about 30 minutes).

In one embodiment, the mixture may optionally be subject to a kineticchallenge. The kinetic challenge helps reduce any non-specific bindingbetween photoaptamers and non-target molecules. In one embodiment, 10 mMdextran sulfate is added and the mixture is incubated for about 15minutes.

In this embodiment, the photoaptamer affinity complexes are convertedinto aptamer covalent complexes by irradiation with light at theappropriate wavelength. For example, irradiation at about 470 nM can beused to crosslink ANA containing photoaptamers to a protein or peptidetarget.

In one embodiment, the catch-2 partition is performed to remove freephotoaptamer. In one embodiment, the mixture containing the aptamercovalent complex is treated with an agent that introduces a capture tagto the target molecule component of the aptamer covalent complexes. Inother embodiments, the tag is introduced before the aptamers arecontacted with the test sample, before the equilibrium binding, orbefore the kinetic challenge. In one embodiment, the target is a proteinor a peptide, and a biotin tag is attached to the target molecule bytreatment with NHS-PEO4-biotin. The mixture is contacted with a solidsupport, the solid support having a capture element adhered to itssurface which is capable of binding to the target capture tag,preferably with high affinity and specificity. In one embodiment, thesolid support is magnetic beads (such as DynaBeads MyOne StreptavidinC1) contained within a well of a microtiter plate and the captureelement is streptavidin. The magnetic beads provide a convenient methodfor the separation of partitioned components of the mixture. Aptamercovalent complexes contained in the mixture are thereby bound to thesolid support through the binding interaction of the target capture tagand capture element. The aptamer covalent complex is partitioned fromthe remainder of the mixture, e.g. by washing the support to removenon-complexed aptamers. In one embodiment, photoaptamer from the aptamercovalent complex can be released for further processing by a methodappropriate for the cleavable moiety. For example, to cleave the PCLinker, the mixture is irradiated with a UV lamp for about 20 minutes.

In another embodiment, the photoaptamer released from the catch-2partition is detected and optionally quantified by any suitable nucleicacid detection method, such as, for example, DNA chip hybridization,Q-PCR, mass spectroscopy, the Invader assay, and the like. Thesedetection methods are described in further detail below. In anotherembodiment the photoaptamer in the aptamer covalent complex is detectedand optionally quantified while still in contact with the solid support.The detected photoaptamer can be correlated with the amount orconcentration of target in the original test sample.

Dual Catch (Catch 1 & 2) Photocrosslinking Assay

In one embodiment, a dual catch photocrosslinking assay, similar to thesingle catch photocrosslinking assay, uses an additional partitioningstep to provide additional sensitivity and specificity (FIG. 2B).

Photoaptamers having high affinity and specificity for a target moleculeare provided. In one embodiment, the crosslinking moiety of thephotoaptamer is linked to the aptamer via a cleavable linker. In oneembodiment, the crosslinking group is ANA (4-azido-2-nitro-aniline) andthe photocleavable group is a PC Linker. In one embodiment, thephotoaptamer also comprises a first releasable tag. In anotherembodiment, the first releasable tag is added at any time in the assayprior to the catch 1 partition. In one embodiment, the first releasabletag moiety is a biotin and the releasable element is a hybridizationlinker. In one embodiment, the photoaptamer construct illustrated inFIG. 6D is used. The photoaptamer is contacted with a sample that maycontain target molecules to form a mixture containing the aptamer, thetarget molecule, and non-target molecules, or sample matrix. Where thetarget molecule is present in the sample, (photo)aptamer affinitycomplexes are formed. The mixture may optionally be incubated for aperiod of time sufficient to achieve equilibrium binding of the aptamersand target molecules (e.g., for at least about 10 minutes, at leastabout 20 minutes, or at least about 30 minutes).

In one embodiment, the mixture may optionally be subject to a kineticchallenge. The kinetic challenge helps reduce any non-specific bindingbetween photoaptamers and non-target molecules. In one particularembodiment, 10 mM dextran sulfate is added and the mixture is incubatedfor about 15 minutes.

(Photo)aptamer affinity complexes are converted into aptamer covalentcomplexes by irradiation with light at the appropriate wavelength. Forexample, irradiation at about 470 nM can be used to crosslink ANAcontaining photoaptamers to a protein target.

In one embodiment, the catch 1 partition is performed to remove freetarget and sample matrix. The mixture is contacted with a first solidsupport having a first capture element adhered to its surface which iscapable of binding to the aptamer capture tag, preferably with highaffinity and specificity. In one embodiment, the first releasable tagcomprises a tag moiety that is a biotin, the first solid support isagarose beads in a column and the first capture element is streptavidin.For example, Pierce Immobilized Streptavidin beads may be used. Aptamercovalent complexes contained in the mixture are thereby bound to thefirst solid support through the binding interaction of the firstreleasable tag and first capture element. The aptamer covalent complexesare partitioned from the remainder of the mixture, e.g. by washing thefirst solid support to remove non-bound molecules.

In one embodiment, aptamer covalent complexes that remain bound to thefirst solid support are then treated with an agent that introduces asecond tag to the target molecule component of the aptamer affinitycomplexes, e.g. biotinylation of a protein or peptide target molecule bytreatment with NHS-PEO4-biotin. The second tag introduced to the targetmolecule may be the same as or different from the first tag. If thesecond tag is the same as the first tag, free capture sites on the firstsolid support may be blocked prior to the initiation of this taggingstep. In this embodiment, the first solid support is washed with freebiotin prior to the initiation of target tagging. Tagging methods, andin particular, tagging of targets such as peptides and proteins aredescribed in detail below. In other embodiments, tagging of the targetis performed at any other point in the assay prior to initiation ofcatch 2 partitioning. If the first and second tags are the same, thetarget is tagged after the capture step of the catch 1 partitioning hasbeen performed.

In another embodiment, the catch 1 partitioning is completed byreleasing the aptamer covalent complexes from the first solid support.In one embodiment, the first releasable tag is a hybridization tagcomplementary to all or some of the first capture element on said solidsupport. This first releasable tag is cleaved by treating the mixturewith conditions that will disrupt the hybridization linker, such as highpH. In one embodiment, 20 mM NaOH is added to the mixture. In otherembodiments, the release of the aptamer covalent complex is accomplishedby any method that is appropriate for the releasable moiety in the firstreleasable tag. Aptamer covalent complexes may be eluted and collectedfor further use in the assay or may be contacted with a further solidsupport in order to conduct the remaining steps of the assay.

In one embodiment, the catch-2 partition is performed to remove freephotoaptamer. The mixture is contacted with a solid support that has, acapture element adhered to its surface which is capable of binding tothe second capture tag, preferably with high affinity and specificity.In one embodiment, the solid support is magnetic beads (such asDynaBeads MyOne Streptavidin C1) contained within a well of a microtiterplate and the capture element is streptavidin. The magnetic beadsprovide a convenient method for the separation of partitioned componentsof the mixture. Aptamer covalent complexes contained in the mixture arethereby bound to the solid support through the binding interaction ofthe target second capture tag and second capture element. The aptamercovalent complex is then partitioned from the remainder of the mixture,e.g. by washing the support to remove non-complexed aptamers. In oneembodiment, photoaptamer from the aptamer covalent complex can then bereleased for further processing by a method appropriate for thecleavable moiety. For example, to cleave the PC Linker, the mixture isirradiated with a UV lamp for about 20 minutes.

In another embodiment, the photoaptamer released from the catch-2partition is detected and optionally quantified by any suitable nucleicacid detection method, such as, for example, DNA chip hybridization,Q-PCR, mass spectroscopy, the Invader assay, and the like. In anotherembodiment the photoaptamer in the aptamer covalent complex is detectedand optionally quantified while still in contact with the solid support.The detected photoaptamer can be correlated with the amount orconcentration of target in the original test sample.

In any of the embodiments disclosed herein, the test sample may beprepared as two or more dilutions of the test sample, which may increasethe dynamic range of target detection by the methods disclosed herein.The individual dilution test samples are separately assayed up to andincluding aptamer (or covalent) complex formation, after which thedilution test samples may be pooled for the remainder of the assay anddetected simultaneously on a single solid support. In one embodiment,each dilution test sample includes a unique aptamer, thereby enabling asingle measurement of the corresponding target. In another embodiment,an aptamer can be added to two or more dilutions, each dilutioncontacting a distinctly tagged aptamer for a particular target, allowingfor the detection of a specific aptamer signal for each of the differentdilution samples on a single solid support. Chaining together dilutedsamples in this manner can extend a dynamic range for a single targetmolecule over many orders of magnitude and add accuracy when overlappingregions of quantification lead to multiple determinations of a singletarget's concentration.

In any of the embodiments disclosed herein, the beads, or solid support,may be suspended after discarding the supernatant containingun-complexed target and test sample or sample matrix. In one embodiment,prior to eluting the free aptamer and aptamer (or covalent) complex fromthe beads and at any point up to suspending the beads, the aptamer (orcovalent) complex may be contacted with a labeling agent, followed byrepeated pelleting and washing to remove unreactive labeling agent priorto contacting the solid support with the aptamer (or covalent) complexfor detection and/or quantification of the target molecule.

In one embodiment, a set of test samples is prepared as serial dilutionsto which a tagged aptamer (or tagged photoaptamer) with a specificaffinity for a target molecule is introduced. The same aptamer with adifferent tag can be added to each test sample dilution. As furtherdescribed herein, following the formation of an aptamer affinity complex(or the optional conversion to an aptamer covalent complex) theindividual test samples can be pooled and contacted with a labelingagent either before or after attachment of the aptamer (or covalent)complex to the solid support. The target molecule, if present in thetest sample, is detected and/or quantified by detecting the labelingagent on the aptamer (or covalent) complex. The resultant signalsdetected for each aptamer having a different tag can be combined toaccurately quantify the amount or concentration of the target moleculein the original test sample. For example, the first dilution may resultin a maximal signal for the target, yielding only semi-quantitativeinformation, while the second dilution may result in a signal that isless than saturating, allowing for an accurate quantification of thetarget in the original test sample.

In another embodiment, a set of test samples is prepared as serialdilutions to which a tagged aptamer (or tagged photoaptamer) with aspecific affinity for a target molecule is introduced. Differentaptamers having unique tags may be added to each sample dilution. Asfurther described herein, following the formation of aptamer affinitycomplexes (or the optional conversion to aptamer covalent complexes) theindividual test samples can be pooled and contacted with a labelingagent either before or after attachment of the aptamer (or covalent)complexes to the solid support. Target molecules present in the testsample are detected and/or quantified by detecting the labeling agent onthe aptamer (or covalent) complex. The resultant signals can bequantified for target ranges over many orders of magnitude depending onthe different serial dilutions of the original sample.

In any of the embodiments disclosed herein, a test sample may becompared to a reference sample. A “reference sample” refers herein toany material, solution, or mixture that contains a plurality ofmolecules and is known to include at least one target molecule. Theprecise amount or concentration of any target molecules present in thereference sample may also be known. The term reference sample includesbiological samples, as defined herein, and samples that may be used forenvironmental or toxicology testing, such as contaminated or potentiallycontaminated water and industrial effluents, for example. A referencesample may also be an end product, intermediate product, or by-productof a preparatory process, for example a manufacturing process. Areference sample may include any suitable assay medium, buffer, ordiluent that has been added to a material, solution, or mixture obtainedfrom an organism or from some other source (e.g., the environment or anindustrial source).

In one embodiment, aptamers with two different probes are prepared. Forexample, one aptamer may have a Cy3 dye and the other the Cy5 dye. Usingthe dual capture crosslinking assay as an example, the reference sampleis exposed to one aptamer and the test sample to the other. Each sampleis separately treated in an identical manner up to and including thecrosslinking step. After crosslinking, the samples can be equally mixedand the remaining steps of the assay may be carried out. A directcomparison of any differential expression (i.e., differential amount orconcentration of the target in the samples) between the reference sampleand the test sample is possible by measuring the signal from eachlabeling agent separately. It should be understood that this method canbe incorporated into any of the other assays describe herein. Further,in addition to using different dyes, including the use of fluorescentdyes, other labels can be employed to differentiate the signal from eachof the different aptamers. For example, in another embodiment, theaptamers used in each of the samples may have different sequence labels.This method is useful, for example, when the readout is Q-PCR or DNAhybridization arrays.

In one embodiment, the reference sample can be a pooled biologicalsample representing a control group. In another embodiment, thereference sample can be a biological sample obtained from an individual,collected at a first time, and the test sample can be obtained from thesame individual but collected at a second time, thereby facilitating alongitudinal study of an individual by measuring and evaluating anychanges in the amount or concentration of one or more target moleculesin multiple biological samples provided by the individual over time.

Any of the methods described herein may be used to conduct a multiplexedanalysis of a test sample. Any such multiplexed analysis can include theuse of at least two, at least tens, at least hundreds, or at leastthousands of aptamers to simultaneously assay an equal number of targetmolecules in a test sample, such as a biological sample, for example. Inthese embodiments, a plurality of aptamers (or labeled photoaptamers,each of which recognizes and optionally cros slinks to a differentanalyte, is introduced to the test sample and any of the above-describedassays can be performed. After release of the aptamers, any suitablemultiplexed nucleic acid detection methods can be employed to measurethe different aptamers that have been released. In one embodiment, thiscan be accomplished by hybridization to complementary probes that areseparately arranged on a solid surface. In another embodiment, each ofthe different aptamers may be detected based on molecular weight usingmass spectroscopy. In yet another embodiment, each of the differentaptamers can be detected based on electrophoretic mobility, such as, forexample, in capillary electrophoresis, in a gel, or by liquidchromatography. In another embodiment, unique PCR probes can be used toquantify each of the different aptamers using Q-PCR.

In each of the assays disclosed herein, a kinetic challenge is used toincrease the specificity of the assay and to reduce non-specificbinding. In one embodiment, which can optionally be employed in each ofthe assays described herein, additional reduction in the non-specificbinding may be accomplished by either preincubation of a competitor withthe test sample or by addition of a competitor to the mixture duringequilibrium binding. In one embodiment, 4 μM of a Z-block competitoroligonucleotide (5′-(ACZZ)₂₈AC-3′, where Z=5-benzyl-dUTP) ispreincubated for about 5 minutes with the test mixture.

Another aspect of the present disclosure relates to kits useful forconveniently performing any of the methods disclosed herein to analyzetest samples. To enhance the versatility of the disclosed methods, thereagents can be provided in packaged combination, in the same orseparate containers, so that the ratio of the reagents provides forsubstantial optimization of the method and assay. The reagents may eachbe in separate containers or various reagents can be combined in one ormore containers depending upon the cross-reactivity and stability of thereagents.

A kit comprises, in packaged combination, at least one tagged aptamerand one or more solid supports, each including at least one captureagent. The kit may also include washing solutions such as bufferedaqueous medium for sample dilution as well as array washing, samplepreparation reagents, and so forth. The kit may further contain reagentsuseful in introducing a second tag, generally through modification orderivatization of the target. In addition the kit may contain reagentssuitable for performing the desired kinetic challenge during theanalytical method. The relative amounts of the various reagents in thekits can be varied widely to provide for concentrations of the reagentsthat substantially optimize the reactions that need to occur during theassay and to further substantially optimize the sensitivity of theassay. Under appropriate circumstances, one or more of the reagents inthe kit can be provided as a dry powder, usually lyophilized, includingexcipients, which upon dissolution will provide a reagent solutionhaving the appropriate concentrations for performing a method or assayin accordance with the present disclosure. The kit can further include awritten description of a method in accordance with any of the methods asdescribed herein.

In one embodiment, a kit for the detection and/or quantification of oneor more target molecules that may be present in a test sample includesat least one aptamer having specific affinity for a target molecule andcomprising a tag; and a solid support, wherein the solid supportincludes at least one capture agent disposed thereon, and wherein thecapture element is capable of associating with the tag on the aptamer.

In another embodiment, a kit for the detection and/or quantification ofone or more target molecules that may be present in a test sampleincludes at least one aptamer having specific affinity for a targetmolecule and comprising a tag and a label; and a solid support, whereinthe solid support includes at least one capture agent disposed thereon,and wherein the capture element is capable of associating with the tagon the aptamer.

In another embodiment, a kit for the detection and/or quantification ofone or more target molecules that may be present in a test sampleincludes at least one aptamer having specific affinity for a targetmolecule and comprising a releasable tag and a label; and a solidsupport, wherein the solid support includes at least one capture agentdisposed thereon, and wherein the capture element is capable ofassociating with the tag on the aptamer.

In addition, any of the above-described kits may contain reagents andmaterials for the performance of a kinetic challenge during thedetection method of the kit.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides of anylength, and such nucleotides may include deoxyribonucleotides,ribonucleotides, and/or analogs or chemically modifieddeoxyribonucleotides or ribonucleotides. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules.

If present, chemical modifications of a nucleotide can include, singlyor in any combination, 2′-position sugar modifications, 5-positionpyrimidine modifications (e.g.,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-napthylmethylcarboxyamide)-2′-deoxyuridine,5-(imidazolylethyl)-2′-deoxyuridine, and5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine), 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo- or 5-iodo-uracil, backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine, and the like.

In one embodiment, the term “C-5 modified pyrimidine” refers to apyrimidine with a modification at the C-5 position including, but notlimited to those moieties illustrated in FIG. 16. Examples of a C-5modified pyrimidine include those described in U.S. Pat. Nos. 5,719,273and 5,945,527. Examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent selected from:benzylcarboxyamide (alternatively benzylaminocarbonyl) (Bn),naphthylmethylcarboxyamide (alternatively naphthylmethylaminocarbonyl)(Nap), tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp),and isobutylcarboxyamide (alternatively isobutylaminocarbonyl) (iBu) asillustrated immediately below.

As delineated above, representative C-5 modified pyrimidines include:5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU) and5-(N-napthylmethylcarboxyamide)-2′-deoxyuridine (NapdU).

Modifications can also include 3′ and 5′ modifications, such as capping.Other modifications can include substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and those with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators, andthose with modified linkages (e.g., alpha anomeric nucleic acids, etc.).Further, any of the hydroxyl groups ordinarily present in a sugar may bereplaced by a phosphonate group or a phosphate group; protected by anysuitable protecting groups; or activated to prepare additional linkagesto additional nucleotides or to a solid support. The 5′ and 3′ terminalOH groups can be phosphorylated or substituted with amines, organiccapping group moieties of from about 1 to about 20 carbon atoms, ororganic capping group moieties of from about 1 to about 20 polyethyleneglycol (PEG) polymers or other hydrophilic or hydrophobic biological orsynthetic polymers. If present, a modification to the nucleotidestructure may be imparted before or after assembly of a polymer. Asequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

In one embodiment, aptamer constructs can include one or moreC5-modified nucleotides in the variable region of the aptamer thateffectively slows the rate of dissociation of the aptamer from itstarget. The base modifications of the nucleotides used in the productionof the variable region of the aptamer have been shown to produceaptamers that have very slow off-rates from their respective targets.For example, there is evidence that an aptamer containing 5 positionmodified pyrimidines, such as any of those illustrated in FIG. 16, hasslow dissociation rate from its target. In some embodiments, the use ofan aptamer that includes nucleotides that have been modified in thismanner in an assay method that includes a kinetic challenge yieldsenhanced sensitivity and specificity in the detection of a target. Asindicated in FIG. 4, aptamers for over 500 targets have been produced todate. Many of these aptamers have slow-off rate characteristics.

In one embodiment, the variable region of the aptamer includesnucleotides that have modified bases. These aptamers may be used in anyof the methods, devices, and kits described herein. There is evidencethat these modified nucleotides may lead to the identification ofaptamers that have very slow off-rates from their respective targetwhile maintaining high affinity to the target. In one embodiment, theC-5 position of the pyrimidine bases may be modified. In otherembodiments, some or all of the pyrimidines in the aptamer may be thebase modified nucleotides. In yet other embodiments, combinations ofmodified pyrimidines may be used. Aptamers containing nucleotides withmodified bases have a number of properties that are different thanaptamers containing only unmodified nucleotides (i.e., naturallyoccurring nucleotides). In one embodiment, the method for modificationof the nucleotides is through a carboxyamide linkage. However othermethods for modification may be suitable. It has been surprisinglyobserved that the structure of the identified slow off-rate aptamers donot appear to conform to the structures predicted by the base pairingmodels. This is supported by the fact that the measured meltingtemperatures of the aptamers are not what the models may predict. Asshown herein, there appears to be little or no correlation between themeasured and predicted melting temperatures. Furthermore, on average,the calculated Tm is 6° C. lower than the measured Tm. The measuredmelting temperatures indicate that aptamers that include these modifiednucleotides are more stable than may be predicted and potentiallypossess novel secondary structures. Identification of slow off-rateaptamers are more likely when modified nucleotides are used in theproduction of the initial library or candidate mixture.

As used herein, “modified nucleic acid” refers to a nucleic acidsequence containing one or more modified nucleotides that are compatiblewith the SELEX process.

In another embodiment of the present disclosure a non-covalent complexof an aptamer and a target is provided, wherein the aptamer has a K_(d)for the target of about 100 nM or less, wherein the rate of dissociation(t_(1/2)) of the aptamer from the target is greater than or equal toabout 30 minutes; is between about 30 minutes and about 240 minutes; is≧ about 30 minutes, ≧ about 60 minutes, ≧ about 90 minutes, ≧ about 120minutes, ≧ about 150 minutes, ≧ about 180 minutes, ≧ about 210 minutes,≧ about 240 minutes; and/or wherein one, several or all pyrimidines inthe nucleic acid sequence of the aptamer are modified at the 5-positionof the base. The modifications may be selected from the group ofcompounds shown in FIG. 16. Aptamers may be designed with anycombination of the base modified nucleotides desired.

As used herein, “aptamer” and “nucleic acid ligand” are usedinterchangeably to refer to a nucleic acid that has a specific bindingaffinity for a target molecule. It is recognized that affinityinteractions are a matter of degree; however, in this context, the“specific binding affinity” of an aptamer for its target means that theaptamer binds to its target generally with a much higher degree ofaffinity than it binds to other components in a test sample. An“aptamer” is a set of copies of one type or species of nucleic acidmolecule that has a particular nucleotide sequence. An aptamer caninclude any suitable number of nucleotides. “Aptamers” refer to morethan one such set of molecules. Different aptamers may have either thesame or different numbers of nucleotides. Any of the methods disclosedherein may include the use of one or more aptamers. Any of the methodsdisclosed herein may also include the use of two or more aptamers thatspecifically bind the same target molecule. As further described below,an aptamer may include a tag. If an aptamer includes a tag, all copiesof the aptamer need not have the same tag. Moreover, if differentaptamers each include a tag, these different aptamers may have eitherthe same tag or a different tag. Aptamers may be ssDNA, dsDNA, RNA or acombination of DNA and RNA.

An aptamer can be identified using any known method, including the SELEXprocess. See, e.g., U.S. Pat. No. 5,475,096 entitled “Nucleic AcidLigands”. Once identified, an aptamer can be prepared or synthesized inaccordance with any known method, including chemical synthetic methodsand enzymatic synthetic methods.

The terms “SELEX” and “SELEX process” are used interchangeably herein torefer generally to a combination of (1) the selection of nucleic acidsthat interact with a target molecule in a desirable manner, for examplebinding with high affinity to a protein, with (2) the amplification ofthose selected nucleic acids. See, e.g., U.S. Pat. No. 5,475,096entitled “Nucleic Acid Ligands”. The SELEX process may be used togenerate an aptamer that covalently binds its target as well as anaptamer that non-covalently binds its target. See, e.g., U.S. Pat. No.5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands byExponential Enrichment Chemi-SELEX”. The SELEX process may also be usedto generate aptamers with improved off-rates as described in U.S.application Ser. No. 12/175,434 entitled “Method for Generating Aptamerswith Improved Off-Rates”, which is being filed concurrently with theinstant application and which is incorporated herein by reference in itsentirety.

As used herein, the term “aptamer affinity complex” or “aptamer complex”refers to a non-covalent complex that is formed by the interaction of anaptamer with its target molecule. An “aptamer affinity complex” or“aptamer complex” is a set of copies of one type or species of complexformed by an aptamer bound to its corresponding target molecule.“Aptamer affinity complexes” or “aptamer complexes” refer to more thanone such set of complexes. An aptamer affinity complex or aptamercomplex can generally be reversed or dissociated by a change in anenvironmental condition, e.g., an increase in temperature, an increasein salt concentration, or an addition of a denaturant.

As used herein, “non-specific complex” refers to a non-covalentassociation between two or more molecules other than an aptamer and itstarget molecule. Because a non-specific complex is not selected on thebasis of an affinity interaction between its constituent molecules, butrepresents an interaction between classes of molecules, moleculesassociated in a non-specific complex will exhibit, on average, muchlower affinities for each other and will have a correspondingly higherdissociation rate than an aptamer and its target molecule. Non-specificcomplexes include complexes formed between an aptamer and a non-targetmolecule, a competitor and a non-target molecule, a competitor and atarget molecule, and a target molecule and a non-target molecule.

The SELEX process generally begins with the preparation of a candidatemixture of nucleic acids of differing sequence. The candidate mixturegenerally includes nucleic acid sequences that include two fixed regions(i.e., each of the members of the candidate mixture contains the samesequences in the same location) and a variable region. Typically, thefixed sequence regions are selected such that they assist in theamplification steps described below, or enhance the potential of a givenstructural arrangement of the nucleic acids in the candidate mixture.The variable region typically provides the target binding region of eachnucleic acid in the candidate mixture, and this variable region can becompletely randomized (i.e., the probability of finding a base at anyposition being one in four) or only partially randomized (e.g., theprobability of finding a base at any location can be selected at anylevel between 0 and 100 percent). The prepared candidate mixture iscontacted with the selected target under conditions that are favorablefor binding to occur between the target and members of the candidatemixture. Under these conditions, the interaction between the target andthe nucleic acids of the candidate mixture generally forms nucleicacid-target pairs that have the strongest relative affinity betweenmembers of the pair. The nucleic acids with the highest affinity for thetarget are partitioned from those nucleic acids with lesser affinity tothe target. The partitioning process is conducted in a manner thatretains the maximum number of high affinity candidates. Those nucleicacids selected during partitioning as having a relatively high affinityto the target are amplified to create a new candidate mixture that isenriched in nucleic acids having a relatively high affinity for thetarget. By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acidmixture to the target will generally increase. Taken to its extreme, theSELEX process will yield a candidate mixture containing one or a verysmall number of unique nucleic acids representing those nucleic acidsfrom the original candidate mixture that have the highest affinity tothe target molecule.

As used herein, “photoaptamer,” “photoreactive nucleic acid ligand,” and“photoreactive aptamer” are used interchangeably to refer to an aptamerthat contains one or more photoreactive functional groups that cancovalently bind to or “crosslink” with a target molecule. For example, anaturally occurring nucleic acid residue may be modified to include achemical functional group that confers photoreactivity upon the nucleicacid residue upon exposure to a radiation source of an appropriatewavelength. A photoaptamer can be identified and/or prepared using anyknown method. In some embodiments, a photoreactive aptamer is identifiedusing the photoSELEX process. See, e.g., U.S. Pat. No. 5,763,177, U.S.Pat. No. 6,001,577, and U.S. Pat. No. 6,291,184, each of which isentitled “Systematic Evolution of Nucleic Acid Ligands by ExponentialEnrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”;see also, e.g., U.S. Pat. No. 6,458,539, entitled “Photoselection ofNucleic Acid Ligands” and U.S. application Ser. No. 12/175,388, entitled“Improved SELEX and PHOTOSELEX”, which is being filed concurrently withthe instant application and which is incorporated herein by reference inits entirety.

Exemplary photoreactive functional groups that may be incorporated intoa photoaptamer include 5-bromouracil, 5-iodouracil, 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-thiouracil,4-thiocytosine, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine.

In addition to these exemplary nucleoside-based photoreactive functionalgroups, other photoreactive functional groups that can be added to aterminal end of an aptamer through an appropriate linker molecule can beused. Such photoreactive functional groups include benzophenone,anthraquinone, 4-azido-2-nitro-aniline, psoralen, derivatives of any ofthese, and the like.

A photoreactive functional group incorporated into a photoaptamer may beactivated by any suitable method. In one embodiment, a photoaptamercontaining a photoreactive functional group is crosslinked to its targetby exposing the photoaptamer affinity complex to a source ofelectromagnetic radiation. Suitable types of electromagnetic radiationinclude ultraviolet light, visible light, X-rays, and gamma rays.Suitable radiation sources include sources that utilize eithermonochromatic light or filtered polychromatic light.

As used herein, the term “aptamer covalent complex” refers to an aptameraffinity complex in which the aptamer has been induced to form orotherwise forms a covalent bond with its target molecule. An “aptamercovalent complex” is a set of copies of one type or species of complexformed by an aptamer covalently bound to its corresponding targetmolecule. “Aptamer covalent complexes” refer to more than one such setof complexes. A covalent bond or linkage between an aptamer and itstarget molecule can be induced by photoactivation of a chemical moietyon the aptamer, including those moieties described above with respect tophotoaptamers. A covalent bond or linkage between an aptamer and itstarget molecule can also be induced chemically. Chemical groups that canbe included in an aptamer and used to induce a covalent linkage with thetarget include but are not limited to aldehydes, maleimides, acrylylderivatives, diazonium derivatives, thiols, etc. In some embodiments,chemical crosslinking groups, such as maleimide or diazonium salts, forexample, can convert aptamer affinity complexes to aptamer covalentcomplexes simply by providing the proper environment and juxtapositionof reactive groups required for specific and sufficiently enhancedchemical reactivity to occur. In other embodiments, chemicalcrosslinkers, such as aldehyde groups, may require the addition ofanother component, for example, sodium cyanoborohydride, to convertaptamer affinity complexes to stable, irreversible aptamer covalentcomplexes. In yet other embodiments, no such chemical crosslinkers areincluded in an aptamer; rather, a third reagent is used to convert anaptamer affinity complex to an aptamer covalent complex by facilitatinga covalent attachment between the aptamer and its target. For example, ahomo- or hetero-bifunctional reagent containing both an amine reactivemoiety (e.g., an N-hydroxy succinimidyl ester, an aldehyde, or animidate) and a nucleoside-reactive group (e.g., an iodoacetamide or anactivated aldehyde) can induce covalent complexation of an aptameraffinity complex, such as an affinity complex formed by an aptamer and atarget protein.

Photoaptamers can be identified by first identifying an affinity aptamerand substituting in one or more photoreactive nucleotide residues.Alternatively, photoaptamers can be identified by a SELEX processcomprising the following: (a) preparing a candidate mixture of nucleicacids, wherein each nucleic acid comprises (i) at least onenon-photoreactive placeholding pyrimidine and (ii) at least one modifiedpyrimidine; (b) contacting the candidate mixture with a target, whereinnucleic acids having an increased affinity to the target relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; (c) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; (d) amplifying the increasedaffinity nucleic acids to yield a nucleic acid ligand-enriched mixtureof nucleic acids, whereby an aptamer to the target compound may beidentified; (e) repeating (b)-(d) as desired; (f) producing a candidatephotoaptamer by replacing in each nucleic acid of the nucleic acidligand-enriched mixture of (d) one or more non-photoreactiveplaceholding pyrimidines with a photoreactive pyrimidine; (g) contactingthe candidate photoaptamer with the target wherein a candidatephotoaptamer-target complex is formed; (h) irradiating said candidatephotoaptamer-target complex; (i) determining whether said candidatephotoaptamer-target complex has photocrosslinked; (j) repeating (f)-(i)as desired; and (k) identifying at least one photoaptamer to the target.

The term “test sample” refers herein to any material, solution, ormixture that contains a plurality of molecules and may include at leastone target molecule. The term test sample includes biological samples,as defined below, and samples that may be used for environmental ortoxicology testing, such as contaminated or potentially contaminatedwater and industrial effluents, for example. A test sample may also bean end product, intermediate product, or by-product of a preparatoryprocess, for example a manufacturing process. A test sample may includeany suitable assay medium, buffer, or diluent that has been added to amaterial, solution, or mixture obtained from an organism or from someother source (e.g., the environment or an industrial source).

The term “biological sample” refers to any material, solution, ormixture obtained from an organism. This includes blood (including wholeblood, leukocytes, peripheral blood mononuclear cells, plasma, andserum), sputum, breath, urine, semen, saliva, meningeal fluid, amnioticfluid, glandular fluid, lymph fluid, nipple aspirate, bronchialaspirate, synovial fluid, joint aspirate, cells, a cellular extract, andcerebrospinal fluid. This also includes experimentally separatedfractions of all of the preceding. The term “biological sample” alsoincludes materials, solutions, or mixtures containing homogenized solidmaterial, such as from a stool sample, a tissue sample, or a tissuebiopsy, for example. The term “biological sample” also includesmaterials, solutions, or mixtures derived from a tissue culture, cellculture, bacterial culture, or viral culture.

As used herein, “target molecule” and “target” are used interchangeablyto refer to any molecule of interest to which an aptamer can bind withhigh affinity and specificity and that may be present in a test sample.A “molecule of interest” includes any minor variation of a particularmolecule, such as, in the case of a protein, for example, minorvariations in amino acid sequence, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent that does not substantially alter the identity of themolecule. A “target molecule” or “target” is a set of copies of one typeor species of molecule or multimolecular structure that is capable ofbinding to an aptamer. “Target molecules” or “targets” refer to morethan one such set of molecules. Exemplary target molecules includeproteins, polypeptides, nucleic acids, carbohydrates, lipids,polysaccharides, glycoproteins, hormones, receptors, antigens,antibodies, affybodies, antibody mimics, viruses, pathogens, toxicsubstances, substrates, metabolites, transition state analogs,cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells,tissues, and any fragment or portion of any of the foregoing. An aptamermay be identified for virtually any chemical or biological molecule ofany size, and thus virtually any chemical or biological molecule of anysize can be a suitable target. A target can also be modified to enhancethe likelihood or strength of an interaction between the target and theaptamer. A target can also be modified to include a tag, as definedabove. In exemplary embodiments, the target molecule is a protein. SeeU.S. Pat. No. 6,376,190 entitled “Modified SELEX Processes WithoutPurified Protein” for methods in which the SELEX target is a peptide.

“Polypeptide,” “peptide,” and “protein” are used interchangeably hereinto refer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Polypeptides can besingle chains or associated chains.

As used herein, “non-target molecule” and “non-target” are usedinterchangeably to refer to a molecule contained in a test sample thatcan form a non-specific complex with an aptamer. A “non-target molecule”or “non-target” is a set of copies of one type or species of molecule ormulti-molecular structure that is capable of binding to an aptamer.“Non-target molecules” or “non-targets” refer to more than one such setof molecules. It will be appreciated that a molecule that is anon-target for a first aptamer may be a target for a second aptamer.Likewise, a molecule that is a target for the first aptamer may be anon-target for the second aptamer.

As used herein, the term “partition” refers to a separation or removalof one or more molecular species from the test sample. Partitioning canbe used to increase sensitivity and/or reduce background. Partitioningis most effective following aptamer complex formation or when theaptamer affinity complex becomes irreversible due to the covalent bondintroduced during crosslinking. A partitioning step may be introducedafter any step, or after every step, where the aptamer affinity complexis immobilized. Partitioning may also rely on a size differential orother specific property that differentially exists between the aptameraffinity complex and other components of the test sample. Partitioningmay also be achieved through a specific interaction with an aptamer ortarget. Partitioning maybe also be accomplished based on the physical orbiochemical properties of the aptamer, target, aptamer affinity complexor aptamer covalent complex

As used herein “Catch 1” refers to the partitioning of an aptameraffinity complex or aptamer covalent complex based on the capture of theaptamer. The purpose of Catch 1 is to remove substantially all of thecomponents in the test sample that are not associated with the aptamer.Removing the majority of such components will generally improve targettagging efficiency by removing non-target molecules from the targettagging step used for Catch 2 capture and may lead to lower assaybackground. In one embodiment, a tag is attached to the aptamer eitherbefore the assay, during preparation of the assay, or during the assayby appending the tag to the aptamer. In one embodiment, the tag is areleasable tag. In one embodiment, the releasable tag comprises acleavable linker and a tag. As described above, tagged aptamer can becaptured on a solid support where the solid support comprises a captureelement appropriate for the tag. The solid support can then be washed toremove any materials in the test mixture that are not associated withthe aptamer.

In various embodiments, aptamer affinity (or covalent) complexes arecaptured or immobilized on the solid support using the capture tagincorporated into the aptamer (aptamer tag). For example, if the capturetag on the aptamer is biotin, as described above, beads having avidin,streptavidin, neutravidin, Extravidin, and like on the surface can beused to capture the aptamer affinity (or covalent) complexes. The beadsare washed to remove any free (uncomplexed) target and other samplematrix components.

In another embodiment, the tag is a hybridization tag complementary to aprobe immobilized on the first solid support. The solid support in thiscase may include microbeads (for example, paramagnetic beads), any othersuitable solid supports described herein, and the like. Thehybridization tag may be a unique sequence tag added to the aptamer orit may be a portion of the aptamer sequence or it may be the entireaptamer sequence. After the aptamer is associated with the solid supportthrough hybridization, the test sample can be washed to remove anymaterials that are not associated with the aptamer. In one embodiment,the aptamer covalent complexes and free aptamer can be released from thesolid support using any suitable method to reverse hybridization, suchas high salt, low or high pH, high temperature or a combination of anyof these. Release of a hybridization tag in Catch 1 is generally notcompatible with preserving aptamer affinity complexes, since theconditions that lead to disruption of tag-probe hybridization willgenerally lead to denaturation of the aptamer's structure, resulting inthe dissociation of the aptamer affinity complex.

In another embodiment, the removal of components not associated with theaptamer can be accomplished using physical techniques rather than anexplicit aptamer tag and a first solid support. In one embodiment, thisis accomplished by precipitating the aptamer, both free and complexed,from the test sample, leaving other molecules that can react with thetarget tagging agent in the supernatant to be discarded. Note that thismethod is designed for use with the photocrosslinking assays. Suchnucleic acid precipitation can be accomplished with reagents thatinclude cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammoniumbromide (DTAB), and organic solvents such as ethanol, for example.

As used herein “Catch 2” refers to the partitioning of an aptameraffinity complex or aptamer covalent complex based on the capture of thetarget molecule. The purpose of the Catch 2 step is to remove free, oruncomplexed, aptamer from the test sample prior to detection andoptional quantification. Removing free aptamer from the sample allowsfor the detection of the aptamer affinity or aptamer covalent complexesby any suitable nucleic acid detection technique. When using Q-PCR fordetection and optional quantification, the removal of free aptamer isneeded for accurate detection and quantification of the target molecule.

In one embodiment, the target molecule is a protein or peptide and freeaptamer is partitioned from the aptamer affinity (or covalent) complex(and the rest of the test sample) using reagents that can beincorporated into proteins (and peptides) and complexes that includeproteins (or peptides), such as, for example, an aptamer affinity (orcovalent) complex. The tagged protein (or peptide) and aptamer affinity(or covalent) complex can be immobilized on a solid support, enablingpartitioning of the protein (or peptide) and the aptamer affinity (orcovalent) complex from free aptamer. Such tagging can include, forexample, a biotin moiety that can be incorporated into the protein orpeptide.

In one embodiment, a Catch 2 tag is attached to the protein (or peptide)either before the assay, during preparation of the assay, or during theassay by chemically attaching the tag to the targets. In one embodimentthe Catch 2 tag is a releasable tag. In one embodiment, the releasabletag comprises a cleavable linker and a tag. It is generally notnecessary, however, to release the protein (or peptide) from the Catch 2solid support. As described above, tagged targets can be captured on asecond solid support where the solid support comprises a capture elementappropriate for the target tag. The solid support can then be washed toremove free aptamer from the solution.

In one embodiment, the target tag introduced for Catch 2 is the same tagas that on the aptamer used for Catch 1. In this embodiment, the targettagging is performed after the Catch 1 step and prior to introduction ofthe Catch 2 solid support. In one embodiment, sites not occupied withaptamers on the Catch 1 support can be blocked prior to tagging targetsif target tagging is done while on the Catch 1 support.

In another embodiment, the aptamer affinity complex or the aptamercovalent complex can be captured on the second solid support directlythrough association with a capture reagent on the second solid support.No explicit target tagging is necessary in this embodiment. In oneembodiment, the second solid support contains an antibody that binds thetarget molecule. In another embodiment, the support contains an Fcfragment that binds the target molecule. In another embodiment, when thetarget molecule is IgG, IgM, IgA or IgE, the support may contain ProteinA to bind the target protein. Any capture reagent that binds to thetarget molecule in an aptamer affinity or aptamer covalent complex canbe used for the Catch 2 step.

In another embodiment, the removal of free aptamer can be accomplishedusing physical techniques rather than an explicit target tag and asecond solid support. In one embodiment where the target molecule is aprotein or peptide, this is accomplished by precipitating the aptamercovalent complexes and leaving free aptamer in the supernatant to bediscarded [note that this works only for covalent complexes]. Suchprotein or peptide precipitation can be accomplished with SDS and highsalt, usually K⁺, for example. After SDS-K⁺ precipitation, the aptamercovalent complex can be recovered for quantification.

After removal of free aptamer by washing the second solid support, theaptamer affinity complexes are then subject to a dissociation step inwhich the complexes are disrupted to yield free aptamer while the targetmolecules generally remain bound to the solid support through thebinding interaction of the probe and target capture tag. The aptamerfrom the aptamer affinity complex can be released by any method thatdisrupts the structure of either the aptamer or the target. This may beachieved though washing of the support bound aptamer affinity complexesin high salt buffer which dissociates the non-covalently boundaptamer-target complexes. Eluted free aptamers are collected anddetected. In another embodiment, high or low pH is used to disrupt theaptamer affinity complexes. In another embodiment high temperature isused to dissociate aptamer affinity complexes. In another embodiment, acombination of any of the above methods may be used.

In the case of aptamer covalent complexes, release of the aptamer forsubsequent quantification is accomplished using a cleavable linker inthe aptamer construct. In another embodiment, a cleavable linker in thetarget tag will result in the release of the aptamer covalent complex.

As used herein, “competitor molecule” and “competitor” are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule, for example to prevent thatnon-target molecule form rebinding non-specifically to an aptamer. A“competitor molecule” or “competitor” is a set of copies of one type orspecies of molecule. “Competitor molecules” or “competitors” refer tomore than one such set of molecules. Competitor molecules includeoligonucleotides, polyanions (e.g., heparin, herring sperm DNA,single-stranded salmon sperm DNA, and polydextrans (e.g., dextransulfate)), abasic phosphodiester polymers, dNTPs, and pyrophosphate. Inthe case of a kinetic challenge that uses a competitor, the competitorcan also be any molecule that can form a non-specific complex with afree aptamer, for example to prevent that aptamer from rebindingnon-specifically to a non-target molecule. Such competitor moleculesinclude polycations (e.g., spermine, spermidine, polylysine, andpolyarginine) and amino acids (e.g., arginine and lysine). When acompetitor is used as the kinetic challenge a fairly high concentrationis utilized relative to the anticipated concentration of total proteinor total aptamer present in the sample. In one embodiment, about 10 mMdextran sulfates is used as the competitor in a kinetic challenge. Inone embodiment, the kinetic challenge comprises adding a competitor tothe mixture containing the aptamer affinity complex, and incubating themixture containing the aptamer affinity complex for a time of greaterthan or equal to about 30 seconds, about 1 minute, about 2 minutes,about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes,about 30 minutes, and about 60 minutes. In another embodiment, thekinetic challenge comprises adding a competitor to the mixturecontaining the aptamer affinity complex and incubating the mixturecontaining the aptamer affinity complex for a time such that the ratioof the measured level of aptamer affinity complex to the measured levelof the non-specific complex is increased.

In some embodiments, the kinetic challenge is performed by diluting thetest sample with binding buffer or any other solution that does notsignificantly increase the natural rate of dissociation of aptameraffinity complexes. The dilution can be about 2×, about 3×, about 4×,about 5×, or any suitable greater dilution. Larger dilutions provide amore effective kinetic challenge by reducing the concentration of totalprotein and aptamer after dilution and, therefore, the rate of theirre-association. If dilution is used to introduce a kinetic challenge,the subsequent test sample mixture containing the aptamer affinitycomplex may be concentrated before further processing. If applicable,this concentration can be accomplished using methods described hereinwith respect to the optional partitioning of any free aptamers from thetest sample and/or the optional removal of other components of the testsample that can react with the tagging agent. When dilution is used asthe kinetic challenge, the amount of dilution is selected to be as highas practical, in view of both the initial test sample volume and thedesirability of recovering the aptamer affinity complex from the final(diluted) volume without incurring a significant loss of the complex. Inone embodiment, the aptamer affinity complex is diluted and the mixtureis incubated for a time ≧ about 30 seconds, ≧ about 1 minute, ≧ about 2minutes, ≧ about 3 minutes, ≧ about 4 minutes, ≧ about 5 minutes, ≧about 10 minutes, ≧ about 30 minutes, and ≧ about 60 minutes. In anotherembodiment, the aptamer affinity complex is diluted and the mixturescontaining the aptamer affinity complex are incubated for a time suchthat the ratio of the measured level of aptamer affinity complex to themeasured level of the non-specific complex is increased.

In some embodiments, the kinetic challenge is performed in such a mannerthat the effect of sample dilution and the effect of introducing acompetitor are realized simultaneously. For example, a test sample canbe diluted with a large volume of competitor. Combining these twokinetic challenge strategies may provide a more effective kineticchallenge than can be achieved using one strategy. In one embodiment,the dilution can be about 2×, about 3×, about 4×, about 5×, or anysuitable greater dilution and the competitor is about 10 mM dextransulfate. In one embodiment, the kinetic challenge comprises diluting themixture containing the aptamer affinity complex, adding a competitor tothe mixture containing the aptamer affinity complex, and incubating themixture containing the aptamer affinity complex for a time greater thanor equal to about 30 seconds, about 1 minute, about 2 minutes, about 3minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 30minutes, and about 60 minutes. In another embodiment, the kineticchallenge comprises diluting the mixture containing the aptamer affinitycomplex, adding a competitor to the mixture containing the aptameraffinity complex and incubating the mixture containing the aptameraffinity complex for a time such that the ratio of the measured level ofaptamer affinity complex to the measured level of the non-specificcomplex is increased.

In various embodiments, aptamer affinity (or covalent) complexes arecaptured or immobilized on the solid support using a tag incorporatedinto the aptamer (aptamer tag), or attached to the target. For example,if the tag on the aptamer is biotin, beads having a capture element suchas avidin, streptavidin, neutravidin, Extravidin, and like on thesurface can be used to capture the aptamer affinity (or covalent)complexes. The beads are washed to remove any free (uncomplexed) target.

As disclosed herein, an aptamer can further comprise a “tag,” whichrefers to a component that provides a means for attaching orimmobilizing an aptamer (and any target molecule that is bound to it) toa solid support. A “tag” is a set of copies of one type or species ofcomponent that is capable of associating with a “capture element”.“Tags” or “capture elements” refers to more than one such set ofcomponents. The tag can be attached to or included in the aptamer by anysuitable method. Generally, the tag allows the aptamer to associate,either directly or indirectly, with a capture element or receptor thatis attached to the solid support. The capture element is typicallychosen (or designed) to be highly specific in its interaction with thetag and to retain that association during subsequent processing steps orprocedures. A tag can enable the localization of an aptamer affinitycomplex (or covalent aptamer affinity complex) to a spatially definedaddress on a solid support. Different tags, therefore, can enable thelocalization of different aptamer covalent complexes to differentspatially defined addresses on a solid support. A tag can be apolynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleicacid, an oligosaccharide, a polysaccharide, an antibody, an affybody, anantibody mimic, a cell receptor, a ligand, a lipid, biotin,polyhistidine, or any fragment or derivative of these structures, anycombination of the foregoing, or any other structure with which acapture element (or linker molecule, as described below) can be designedor configured to bind or otherwise associate with specificity.Generally, a tag is configured such that it does not interactintramolecularly with either itself or the aptamer to which it isattached or of which it is a part. If SELEX is used to identify anaptamer, the tag may be added to the aptamer either pre- or post-SELEX.In one embodiment, the tag is included on the 5′-end of the aptamerpost-SELEX. In another embodiment, the tag is included on the 3′-end ofthe aptamer post-SELEX. In yet another embodiment, tags may be includedon both the 3′ and 5′ ends of the aptamers in a post-SELEX modificationprocess. In another embodiment, the tag may be an internal segment ofthe aptamer.

In one embodiment, the tag is a biotin group and the capture element isa biotin binding protein such as avidin, streptavidin, neutravidin,Extravidin. This combination is may be conveniently used in variousembodiments, as biotin is easily incorporated into aptamers duringsynthesis and streptavidin beads are readily available.

In one embodiment, the tag is polyhistidine and the capture element isnitrilotriacetic acid (NTA) chelated with a metal ion such as nickel,cobalt, iron, or any other metal ion able to form a coordinationcompound with poly-histidine when chelated with NTA.

In one embodiment, the tag is a polynucleotide that is designed tohybridize directly with a capture element that contains a complementarypolynucleotide sequence. In this case, the tag is sometimes referred toas a “sequence tag” and the capture element is generally referred to asa “probe”. In this embodiment, the tag is generally configured and thehybridization reaction is carried out under conditions such that the tagdoes not hybridize with a probe other than the probe for which the tagis a perfect complement. This allows for the design of a multiplex assayformat as each tag/probe combination can have unique sequences.

In some embodiments, the tag comprises nucleotides that are a part ofthe aptamer itself. For example, if SELEX is used to identify anaptamer, the aptamer generally includes a 5′-fixed end separated from a3′-fixed end by a nucleotide sequence that varies, depending upon theaptamer, that is, a variable region. In one embodiment, the tag cancomprise any suitable number of nucleotides included in a fixed end ofthe aptamer, such as, for example, an entire fixed end or any portion ofa fixed end, including nucleotides that are internal to a fixed end. Inanother embodiment, the tag can comprise any suitable number ofnucleotides included within the variable region of the aptamer, such as,for example, the entire variable region or any portion of the variableregion. In a further embodiment, the tag can comprise any suitablenumber of nucleotides that overlap both the variable region and one ofthe fixed ends, that is, the tag can comprise a nucleotide sequence thatincludes any portion (including all) of the variable region and anyportion (including all) of a fixed end.

In another embodiment, a tag can associate directly with a probe andcovalently bind to the probe, thereby covalently linking the aptamer tothe surface of the solid support. In this embodiment, the tag and theprobe can include suitable reactive groups that, upon association of thetag with the probe, are sufficiently proximate to each other to undergoa chemical reaction that produces a covalent bond. The reaction mayoccur spontaneously or may require activation, such as, for example,photo-activation or chemical activation. In one embodiment, the tagincludes a diene moiety and the probe includes a dienophile, andcovalent bond formation results from a spontaneous Diels-Alderconjugation reaction of the diene and dienophile. Any appropriatecomplementary chemistry can be used, such as, for example, N-Mannichreaction, disulfide formation, Curtius reaction, Aldol condensation,Schiff base formation, and Michael addition.

In another embodiment, the tag associates indirectly with a probe, suchas, for example, through a linker molecule, as further described below.In this embodiment, the tag can include a polynucleotide sequence thatis complementary to a particular region or component of a linkermolecule. The tag is generally configured and the hybridization reactionis carried out such that the tag does not hybridize with apolynucleotide sequence other than the polynucleotide sequence includedin the linker molecule.

If the tag includes a polynucleotide, the polynucleotide can include anysuitable number of nucleotides. In one embodiment, a tag includes atleast about 10 nucleotides. In another embodiment, the tag includes fromabout 10 to about 45 nucleotides. In yet another embodiment, the tagincludes at least about 30 nucleotides. Different tags that include apolynucleotide can include either the same number of nucleotides or adifferent number of nucleotides.

In some embodiments, the tag component is bi-functional in that itincludes functionality for specific interaction with a capture elementon a solid support or “probe” as defined below (probe associationcomponent), and functionality for dissociating the molecule to which itis attached from the probe association component of the tag. The meansfor dissociating the probe association component of the tag includeschemical means, photochemical means or other means depending upon theparticular tag that is employed.

In some embodiments, the tag is attached to the aptamer. In otherembodiments, the tag is attached to the target molecule. The tag may beattached to the target molecule prior to the aptamer binding step, ormay be attached to the target molecule or aptamer affinity (or covalent)complex after binding equilibration (or photo-crosslinking) has beenachieved.

As used herein, “capture element”, “probe” or “receptor” refers to amolecule that is configured to associate, either directly or indirectly,with a tag. A “capture element”, “probe” or “receptor” is a set ofcopies of one type of molecule or one type of multi-molecular structurethat is capable of immobilizing the moiety to which the tag is attachedto a solid support by associating, either directly or indirectly, withthe tag. “Capture elements” “probes” or “receptors” refer to more thanone such set of molecules. A capture element, probe or receptor can be apolynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleicacid, an oligosaccharide, a polysaccharide, an antibody, an affybody, anantibody mimic, a cell receptor, a ligand, a lipid, biotin,polyhistidine, or any fragment or derivative of these structures, anycombination of the foregoing, or any other structure with which a tag(or linker molecule) can be designed or configured to bind or otherwiseassociate with specificity. A capture element, probe or receptor can beattached to a solid support either covalently or non-covalently by anysuitable method.

While the terms “capture element”, “probe” and “receptor” are usedinterchangeably, probe generally refers to a polynucleotide sequence. Inone embodiment, the probe includes a polynucleotide that has a sequencethat is complementary to a polynucleotide tag sequence. In thisembodiment, the probe sequence is generally configured and thehybridization reaction is carried out under conditions such that theprobe does not hybridize with a nucleotide sequence other than the tagfor which the probe includes the complementary sequence (i.e., the probeis generally configured and the hybridization reaction is carried outunder conditions such that the probe does not hybridize with a differenttag or an aptamer).

In another embodiment, the probe associates indirectly with a tag, forexample, through a linker molecule. In this embodiment, the probe caninclude a polynucleotide sequence that is complementary to a particularregion or component of a linker molecule. The probe is generallyconfigured and the hybridization reaction is carried out such that theprobe does not hybridize with a polynucleotide sequence other than thepolynucleotide sequence included in the linker molecule.

If a probe includes a polynucleotide, the polynucleotide can include anysuitable number of nucleotides. In one embodiment, a probe includes atleast about 10 nucleotides. In another embodiment, a probe includes fromabout 10 to about 45 nucleotides. In yet another embodiment, a probeincludes at least about 30 nucleotides. Different probes that include apolynucleotide can include either the same number of nucleotides or adifferent number of nucleotides.

In some embodiments, the capture probe is bi-functional in that itincludes functionality for specific interaction with a polynucleotidetag, and functionality for dissociating the probe from the solid supportsuch that the probe and aptamer are simultaneously released. The meansfor dissociating the probe from the solid support includes chemicalmeans, photochemical means or other means depending upon the particularcapture probe that is employed.

Due to the reciprocal nature of the interaction between a particular tagand capture element pair, a tag in one embodiment may be used as acapture element in another embodiment, and a capture element in oneembodiment may be used as a tag in another embodiment. For example, anaptamer with a biotin tag may be captured with streptavidin attached toa solid support in one embodiment, while an aptamer with a streptavidintag may be captured with biotin attached to a solid support in anotherembodiment.

In some embodiments, it is desirable to immobilize aptamer affinity (orcovalent) complexes to a solid support to enable the isolation ofaptamer affinity (or covalent) complexes and remove free aptamer. In oneembodiment, the tag is added to the target molecule of the affinity (orcovalent) complex using a reagent that is highly reactive with thetarget molecule and weakly reactive (or ideally non-reactive) with theaptamer. In this embodiment, the tag is designed such that targettagging of aptamer affinity complexes is accomplished with little or nodissociation of the affinity complex, for example, at a pH and ionicstrength that is compatible with the affinity interaction and does notchange the conformation of the target or the aptamer, and a degree oftagging that does not introduce a large plurality of tags on each targetmolecule such that interaction with the aptamer is affected. Targettagging of aptamer covalent complexes does not share these restrictionsand can be accomplished under any conditions suitable for efficienttagging.

In some embodiments, it may be important to assure that the taggingreagent tags most, if not all, proteins present in a test sample buttends not to tag, or tags only minimally, nucleic acids or othercomponents of the assay, such as the solid support. Any reactivechemical group found on proteins, but not found on nucleic acids or thesubstrate surface, can serve as a site of covalent attachment. Exemplaryreactive chemical groups include primary amines (e.g., on lysineresidues), thiols (e.g., on cysteine, which may be produced by thereduction of disulfide linkages), alcohols (e.g., on serine, threonine,tyrosine, and sugar moieties on glycoproteins (including the products ofoxidation of cis-diols on such sugars)), and carboxylates (e.g., onglutamic and aspartic acid). In one embodiment, the tagging reagentcomprises an N-hydroxysuccinimide-activated tag, which reactspreferentially with lysine residues on proteins and peptides.

The optimal conditions for tagging different aptamer affinity (orcovalent) complexes may be different, and are normally determinedempirically to optimize the sensitivity of the method. In oneembodiment, the concentration of the tagging agent is usually sufficientto detect at least about 1% of the target molecules. In anotherembodiment, the concentration of the labeling agent is usuallysufficient to detect at least about 10% of the target molecules. In afurther embodiment, the concentration of the tagging agent is usuallysufficient to detect at least about 90% of the target molecules.

In one embodiment the target is a protein or peptide and the tag is abiotin attached to the target using a standard reagent for proteinbiotinylation, such as, for example, NHS-PEO4-biotin. Other suitablereagents include Sulfo-NHS-LC-biotin, PFP-biotin, TFP-PEO₃-biotin, orany other suitable reagent that may be used to attach a tag to aprotein.

As used herein, a linker is a molecular structure that is use to connecttwo functional groups or molecular structures. As used herein, “spacinglinker” or more simply a “spacer” refers to a group of benign atoms thatprovide separation or spacing between two different functional groupswithin an aptamer. As used herein, a “releasable” or “cleavable”element, moiety, or linker refers to a molecular structure that can bebroken to produce two separate components. A releasable (or cleavable)element may comprise a single molecule in which a chemical bond can bebroken (referred to herein as an “inline cleavable linker”), or it maycomprise two or more molecules in which a non-covalent interaction canbe broken or disrupted (referred to herein as a “hybridization linker”).

In some embodiments, it necessary to spatially separate certainfunctional groups from others in order to prevent interference with theindividual functionalities. For example, the presence of a label, whichabsorbs certain wavelengths of light, proximate to a photocleavablegroup can interfere with the efficiency of photocleavage. It istherefore desirable to separate such groups with a non-interferingmoiety that provides sufficient spatial separation to recover fullactivity of photocleavage, for example. In some embodiments, a “spacinglinker” has been introduced into an aptamer with both a label andphotocleavage functionality.

In one embodiment, spacing linkers are introduced into the aptamerduring synthesis and so can be comprised of number of phosphoramiditespacers, including but limited to aliphatic carbon chains of length 3,6, 9, 12 and 18 carbon atoms, polyethylene glycol chains of length 1, 3,and 9 ethylene glycol units, or a tetrahydrofuran moiety (termed dSpacer(Glenn Research) or any combination of the foregoing or any otherstructure or chemical component that can be designed or configured toadd length along a phosphodiester backbone. In another embodiment, thespacing linker includes polynucleotides, such as poly dT, dA, dG, or dCor poly U, A, G, or C or any combination of the foregoing. In anotherembodiment, spacers include one or more abasic ribose or deoxyribosemoieties. Note that such sequences are designed such that they do notinterfere with the aptamer's structure or function.

As used herein, an “inline cleavable linker” refers to a group of atomsthat contains a releasable or cleavable element. In some embodiments, aninline cleavable linker is used to join an aptamer to a tag, therebyforming a releasable tag. For example, an inline releasable linker canbe utilized in any of the described assays to create a releasableconnection between an aptamer and a biotin (e.g., in the affinity assaysand cros slinking assays) or a releasable connection between an aptamerand a photocrosslinking group (e.g. in the crosslinking assays).

In one embodiment, the inline cleavable linker may be photo-cleavable inthat it includes a bond that can be cleaved by irradiating thereleasable element at the appropriate wavelength of light. In anotherembodiment, the inline cleavable linker may be chemically cleavable inthat it includes a bond that can be cleaved by treating it with anappropriate chemical or enzymatic reagent. In another embodiment, thereleasable element includes a disulfide bond that can be cleaved bytreating it with a reducing agent to disrupt the bond.

As used herein, a “hybridization linker” refers to a linker thatcomprises two or more molecules in which a non-covalent interaction canbe broken or disrupted through chemical or physical methods. In someembodiments, a hybridization linker is used to join an aptamer to a tag,thereby forming a releasable tag. For example, a hybridization linkercan be utilized in any of the described assays to create a releasableconnection between an aptamer and a biotin (e.g. in the affinity assaysand crosslinking assays) or a releasable connection between an aptamerand a photocrosslinking group (e.g. in the crosslinking assays).

In one embodiment, a hybridization linker comprises two nucleic acidsthat hybridize to form a non-covalent bond. In one embodiment, one ofthe nucleic acids that forms the hybridization link can be a region ofthe aptamer itself and the other nucleic acid can be a nucleic acid thatis complementary to that region. Release can be accomplished by anysuitable mechanism for disrupting nucleic acid duplexes (while stillmaintaining compatibility with the assay). In one embodiment, 20 mM NaOHis used to disrupt the hybridization linker in the dual catchphotocrosslinking assay. A hybridization linker molecule may have anysuitable configuration and can include any suitable components,including one or more polynucleotides, polypeptides, peptide nucleicacids, locked nucleic acids, oligosaccharides, polysaccharides,antibodies, affybodies, antibody mimics or fragments, receptors,ligands, lipids, any fragment or derivative of these structures, anycombination of the foregoing, or any other structure or chemicalcomponent that can be designed or configured to form a releasablestructure.

In one embodiment, the releasable tag consists of at least onepolynucleotide consisting of a suitable number of nucleotides. In oneembodiment, a polynucleotide component of a linker molecule includes atleast about 10 nucleotides. In another embodiment, a polynucleotidecomponent of a linker molecule includes from about 10 to about 45nucleotides. In yet another embodiment, a polynucleotide component of alinker molecule includes at least about 30 nucleotides. Linker moleculesused in any of the methods disclosed herein can include polynucleotidecomponents having either the same number of nucleotides or a differentnumber of nucleotides.

“Solid support” refers to any substrate having a surface to whichmolecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The solid support may include anysubstrate material that is capable of providing physical support for thecapture elements or probes that are attached to the surface. Thematerial is generally capable of enduring conditions related to theattachment of the capture elements or probes to the surface and anysubsequent treatment, handling, or processing encountered during theperformance of an assay. The materials may be naturally occurring,synthetic, or a modification of a naturally occurring material. Suitablesolid support materials may include silicon, a silicon wafer chip,graphite, mirrored surfaces, laminates, membranes, ceramics, plastics(including polymers such as, e.g., poly(vinyl chloride), cyclo-olefincopolymers, agarose gels or beads, polyacrylamide, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene(PTFE or Teflon®), nylon, poly(vinyl butyrate)), germanium, galliumarsenide, gold, silver, Langmuir Blodgett films, a flow through chip,etc., either used by themselves or in conjunction with other materials.Additional rigid materials may be considered, such as glass, whichincludes silica and further includes, for example, glass that isavailable as Bioglass. Other materials that may be employed includeporous materials, such as, for example, controlled pore glass beads,crosslinked beaded Sepharose® or agarose resins, or copolymers ofcrosslinked bis-acrylamide and azalactone. Other beads include polymerbeads, solid core beads, paramagnetic beads, or microbeads. Any othermaterials known in the art that are capable of having one or morefunctional groups, such as any of an amino, carboxyl, thiol, or hydroxylfunctional group, for example, incorporated on its surface, are alsocontemplated.

The material used for a solid support may take any of a variety ofconfigurations ranging from simple to complex. The solid support canhave any one of a number of shapes, including a strip, plate, disk, rod,particle, bead, tube, well (microtiter), and the like. The solid supportmay be porous or non-porous, magnetic, paramagnetic, or non-magnetic,polydisperse or monodisperse, hydrophilic or hydrophobic. The solidsupport may also be in the form of a gel or slurry of closely-packed (asin a column matrix) or loosely-packed particles.

In one embodiment, the solid support with attached capture element isused to capture tagged aptamer affinity complexes or aptamer covalentcomplexes from a test mixture. In one particular example, when the tagis a biotin moiety, the solid support could be a streptavidin-coatedbead or resin such as Dynabeads M-280 Streptavidin, Dynabeads MyOneStreptavidin, Dynabeads M-270 Streptavidin (Invitrogen), StreptavidinAgarose Resin (Pierce), Streptavidin Ultralink Resin, MagnaBindStreptavidin Beads (ThermoFisher Scientific), BioMag Streptavidin,ProMag Streptavidin, Silica Streptavidin (Bangs Laboratories),Streptavidin Sepharose High Performance (GE Healthcare), StreptavidinPolystyrene Microspheres (Microspheres-Nanospheres), Streptavidin CoatedPolystyrene Particles (Spherotech), or any other streptavidin coatedbead or resin commonly used by one skilled in the art to capturebiotin-tagged molecules.

As has been described above, one object of the instant invention is toconvert a protein signal into an aptamer signal. As a result thequantity of aptamers collected/detected is indicative of, and may bedirectly proportional to, the quantity of target molecules bound and tothe quantity of target molecules in the sample. A number of detectionschemes can be employed without eluting the aptamer affinity or aptamercovalent complex from the second solid support after Catch 2partitioning. In addition to the following embodiments of detectionmethods, other detection methods will be known to one skilled in theart.

Many detection methods require an explicit label to be incorporated intothe aptamer prior to detection. In these embodiments, labels, such as,for example, fluorescent or chemiluminescent dyes can be incorporatedinto aptamers either during or post synthesis using standard techniquesfor nucleic acid synthesis. Radioactive labels can be incorporatedeither during synthesis or post synthesis using standard enzymereactions with the appropriate reagents. Labeling can also occur afterthe Catch 2 partitioning and elution by using suitable enzymatictechniques. For example, using a primer with the above mentioned labels,PCR will incorporate labels into the amplification product of the elutedaptamers. When using a gel technique for quantification, different sizemass labels can be incorporated using PCR as well. These mass labels canalso incorporate different fluorescent or chemiluminescent dyes foradditional multiplexing capacity. Labels may be added indirectly toaptamers by using a specific tag incorporated into the aptamer, eitherduring synthesis or post synthetically, and then adding a probe thatassociates with the tag and carries the label. The labels include thosedescribed above as well as enzymes used in standard assays forcolorimetric readouts, for example. These enzymes work in combinationwith enzyme substrates and include enzymes such as, for example,horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels mayalso include materials or compounds that are electrochemical functionalgroups for electrochemical detection.

For example, the aptamer may be labeled, as described above, with aradioactive isotope such as ³²P prior to contacting the test sample.Employing any one of the four basic assays, and variations thereof asdiscussed above, aptamer detection may be simply accomplished byquantifying the radioactivity on the second solid support at the end ofthe assay. The counts of radioactivity will be directly proportional tothe amount of target in the original test sample. Similarly, labeling anaptamer with a fluorescent dye, as described above, before contactingthe test sample allows for a simple fluorescent readout directly on thesecond solid support. A chemiluminescent label or a quantum dot can besimilarly employed for direct readout from the second solid support,requiring no aptamer elution.

By eluting the aptamer or releasing photoaptamer covalent complex fromthe second solid support additional detection schemes can be employed inaddition to those described above. For example, the released aptamer,photoaptamer or photoaptamer covalent complex can be run on a PAGE geland detected and optionally quantified with a nucleic acid stain, suchas SYBR Gold. Alternatively, the released aptamer, photoaptamer orphotoaptamer covalent complex can be detected and quantified usingcapillary gel electrophoresis (CGE) using a fluorescent labelincorporated in the aptamer as described above. Another detection schemeemploys quantitative PCR to detect and quantify the eluted aptamer usingSYBR Green, for example. Alternatively, the Invader® DNA assay may beemployed to detect and quantify the eluted aptamer.

In another embodiment, the amount or concentration of the aptameraffinity complex (or aptamer covalent complex) is determined using a“molecular beacon” during a replicative process (see, e.g., Tyagi etal., Nat. Biotech. 16:49 53, 1998; U.S. Pat. No. 5,925,517). A molecularbeacon is a specific nucleic acid probe that folds into a hairpin loopand contains a fluorophore on one end and a quencher on the other end ofthe hairpin structure such that little or no signal is generated by thefluorophore when the hairpin is formed. The loop sequence is specificfor a target polynucleotide sequence and, upon hybridizing to theaptamer sequence the hairpin unfolds and thereby generates a fluorescentsignal.

For multiplexed detection of a small number of aptamers still bound tothe second solid support, fluorescent dyes with differentexcitation/emission spectra can be employed to detect and quantify two,or three, or five, or up to ten individual aptamers. Similarly differentsized quantum dots can be employed for multiplexed readouts. The quantumdots can be introduced after partitioning free aptamer from the secondsolid support. By using aptamer specific hybridization sequencesattached to unique quantum dots multiplexed readings for 2, 3, 5, and upto 10 aptamers can be performed. Labeling different aptamers withdifferent radioactive isotopes that can be individually detected, suchas ³²P, ¹²⁵I, ³H, ¹³C, and ³⁵S, can also be used for limited multiplexreadouts.

For multiplexed detection of aptamers released from the Catch 2 secondsolid support, a single fluorescent dye, incorporated into each aptameras described above, can be used with a quantification method that allowsfor the identification of the aptamer sequence along with quantificationof the aptamer level. Methods include but are not limited to DNA chiphybridization, micro-bead hybridization, and CGE analysis.

In one embodiment, a standard DNA hybridization array, or chip, is usedto hybridize each aptamer or photoaptamer to a unique or series ofunique probes immobilized on a slide or chip such as Agilent arrays,Illumina BeadChip Arrays, or NimbleGen arrays. Each unique probe iscomplementary to a sequence on the aptamer. The complementary sequencemay be a unique hybridization tag incorporated in the aptamer, or aportion of the aptamer sequence, or the entire aptamer sequence. Theaptamers released from the Catch 2 solid support are added to anappropriate hybridization buffer and processed using standardhybridization methods. For example, the aptamer solution is incubatedfor 12 hours with a DNA hybridization array at about 60° C. to ensurestringency of hybridization. The arrays are washed and then scanned in afluorescent slide scanner, producing an image of the aptamerhybridization intensity on each feature of the array. Image segmentationand quantification is accomplished using image processing software, suchas ArrayVision. In one embodiment, multiplexed aptamer assays can bedetected using up to 25 aptamers, up to 50 aptamers, up to 100 aptamers,up to 200 aptamers, up to 500 aptamers, up to 1000 aptamers, and up to10,000 aptamers.

In one embodiment, addressable micro-beads having unique DNA probescomplementary to the aptamers as described above are used forhybridization. The micro-beads may be addressable with uniquefluorescent dyes, such as Luminex beads technology, or use bar codelabels as in the Illumina VeraCode technology, or laser poweredtransponders. In one embodiment, the aptamers released from the Catch 2solid support are added to an appropriate hybridization buffer andprocessed using standard micro-bead hybridization methods. For example,the aptamer solution is incubated for two hours with a set ofmicro-beads at about 60° C. to ensure stringency of hybridization. Thesolutions are then processed on a Luminex instrument which counts theindividual bead types and quantifies the aptamer fluorescent signal. Inanother embodiment, the VeraCode beads are contacted with the aptamersolution and hybridized for two hours at about 60° C. and then depositedon a gridded surface and scanned using a slide scanner foridentification and fluorescence quantification. In another embodiment,the transponder micro-beads are incubated with the aptamer sample atabout 60° C. and then quantified using an appropriate device for thetransponder micro-beads. In one embodiment, multiplex aptamer assays canbe detected by hybridization to micro-beads using up to 25 aptamers, upto 50 aptamers, up to 100 aptamers, up to 200 aptamers, and up to 500aptamers.

The sample containing the eluted aptamers can be processed toincorporate unique mass tags along with fluorescent labels as describedabove. The mass labeled aptamers are then injected into a CGEinstrument, essentially a DNA sequencer, and the aptamers are identifiedby their unique masses and quantified using fluorescence from the dyeincorporated during the labeling reaction. One exemplary example of thistechnique has been developed by Althea Technologies.

In many of the methods described above, the solution of aptamers can beamplified and optionally tagged before quantification. Standard PCRamplification can be used with the solution of aptamers eluted from theCatch 2 solid support. Such amplification can be used prior to DNA arrayhybridization, micro-bead hybridization, and CGE readout.

In another embodiment, the aptamer affinity complex (or aptamer covalentcomplex) is detected and/or quantified using Q-PCR. As used herein,“Q-PCR” refers to a PCR reaction performed in such a way and under suchcontrolled conditions that the results of the assay are quantitative,that is, the assay is capable of quantifying the amount or concentrationof aptamer present in the test sample.

In one embodiment, the amount or concentration of the aptamer affinitycomplex (or aptamer covalent complex) in the test sample is determinedusing TaqMan® PCR. This technique generally relies on the 5′-3′exonuclease activity of the oligonucleotide replicating enzyme togenerate a signal from a targeted sequence. A TaqMan probe is selectedbased upon the sequence of the aptamer to be quantified and generallyincludes a 5′-end fluorophore, such as 6-carboxyfluorescein, forexample, and a 3′-end quencher, such as, for example, a6-carboxytetramethylfluorescein, to generate signal as the aptamersequence is amplified using polymerase chain reaction (PCR). As thepolymerase copies the aptamer sequence, the exonuclease activity freesthe fluorophore from the probe, which is annealed downstream from thePCR primers, thereby generating signal. The signal increases asreplicative product is produced. The amount of PCR product depends uponboth the number of replicative cycles performed as well as the startingconcentration of the aptamer.

In another embodiment, the amount or concentration of an aptameraffinity complex (or aptamer covalent complex) is determined using anintercalating fluorescent dye during the replicative process. Theintercalating dye, such as, for example, SYBR® green, generates a largefluorescent signal in the presence of double-stranded DNA as compared tothe fluorescent signal generated in the presence of single-stranded DNA.As the double-stranded DNA product is formed during PCR, the signalproduced by the dye increases. The magnitude of the signal produced isdependent upon both the number of PCR cycles and the startingconcentration of the aptamer.

In another embodiment, the aptamer affinity complex (or aptamer covalentcomplex) is detected and/or quantified using mass spectrometry. Uniquemass tags can be introduced using enzymatic techniques described above.For mass spectroscopy readout, no detection label is required, ratherthe mass itself is used to both identify and, using techniques commonlyused by those skilled in the art, quantified based on the location andarea under the mass peaks generated during the mass spectroscopyanalysis. An example using mass spectroscopy is the MassARRAY® systemdeveloped by Sequenom.

In other embodiments, aptamer constructs that include different built-infunctionalities are provided. These functionalities may include tags forimmobilization, labels for detection, photoreactive groups, means topromote or control separation, etc. In one embodiment, an aptamerincludes a cleavable or releasable section (also described as an elementor component) in the aptamer sequence. These additional components orelements are structural elements or components that introduce additionalfunctionality into the aptamer. In other embodiments, the aptamerincludes one or more of the following additional components (alsodescribed as a functional or structural element or component or moiety):a labeled or detectable component, a spacer component, a cleavableelement, and a specific binding tag or immobilization element orcomponent. For example, in one embodiment of the photocrosslinkingaptamer, the aptamer includes a tag connected to the aptamer via acleavable moiety, a label, a spacer component separating the label andthe cleavable moiety, and a photocrosslinking moiety, as shown in FIG.3L.

All aptamer constructs can be synthesized using standard phosphoramiditechemistry. Representative aptamer constructs are shown in FIG. 3Athrough FIG. 3L. The functionality can be split between the 5′ and 3′end or combined on either end. In addition to photocleavable moieties,other cleavable moieties can be used, including chemically orenzymatically cleavable moieties. A variety of spacer moieties can beused and one or more biotin moieties can be included. Tags (alsoreferred to as immobilization or specific binding elements orcomponents) other than biotin can also be incorporated. Suitableconstruction reagents include biotin phosphoramidite, PC Linker (GlenResearch PN 10-4920-02); PC biotin phosphoramidite (Glen Research PN10-4950-02); dSpacer CE phosphoramidite (Glen Research PN 10-1914-02);Cy3 phosphoramidite (Glen Research PN 10-5913-02); and Arm26-Ach SpacerAmidite (Fidelity Systems PN SP26Ach-05). As illustrated in FIG. 3K, afluorescent dye (such as Cy3), a spacer, the photocleavable and biotinmoieties may be added to the end of the aptamer. In one embodiment,because of potential interactions between the photocleavable moiety andthe dye, the spacer is inserted between these two moieties.

In one embodiment, the tag is covalently attached to the aptamer, asillustrated in FIGS. 3I-3K. In another embodiment, the tag is indirectlyattached to the aptamer in the form of a hybridized polynucleotidesequence, which is complementary to a portion of the aptamer sequence,that has a covalently attached tag, as illustrated in FIGS. 3E-3H.

In one embodiment, an aptamer can be further modified to include a firstcleavable moiety positioned between the crosslinking group and a uniquesequence within the aptamer. This first cleavable moiety may, forexample, be cleavable by a variety of different means including,chemical, photochemical or ionic depending upon the cleavable moietyemployed. In one embodiment, an aptamer has the structure is shown inFIG. 3B. For example, the photocrosslinking group may be4-azido-2-nitro-aniline and the photocleavable group, or releasablemoiety, may be a PC Linker available from Glen Research as aphosphoramidite(-(4,4′-dimethoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite).

In other embodiments, the aptamer includes a capture tag that is joinedto the aptamer through a releasable group. For example, a biotin capturetag may be attached to the aptamer via a second oligonucleotide thathybridizes to the aptamer as illustrated in FIG. 3F. In otherembodiments, other capture tags or cleavable elements can be attached tothe same aptamer. For example, a poly-His tag may be attached to theaptamer via a second chemical or photocleavable moiety. The advantage tothis aptamer construct is that two different processing steps can beapplied to separate the aptamer affinity (or covalent) complex fromother components in the test sample. These separation steps can be usedin any sequence desired.

In another embodiment, a detection label can also be included within theaptamer as illustrated in FIG. 3C. This detection label provides for thedetection and/or quantification of the free aptamer in the final step ofthe assay as described in detail above. For example, for fluorescentdetection, a fluorescent dye such as Cy3 or Cy5 dye may be incorporatedwithin aptamer. A Cy3 may be introduced using the Cy3 phosphoramiditefrom Glen Research([344-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidityl]propyl]-3,3,3′,3′-tetramethylindocarbocyaninechloride)), but any suitable label may be included in the aptamer.

As used in this specification, including the appended claims, thesingular forms “a,” “an,” and “the” include plural references, unlessthe content clearly dictates otherwise, and are used interchangeablywith “at least one” and “one or more.” Thus, reference to “an aptamer”includes mixtures of aptamers, reference to “a probe” includes mixturesof probes, and the like.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical values such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, “associate,” “associates,” and any variation thereofrefers to an interaction or complexation between a tag and a proberesulting in a sufficiently stable complex so as to permit separation of“unassociated” or unbound materials, such as, for example, unboundcomponents of a test sample, from the tag-probe complex under givencomplexation or reaction conditions. A tag and a probe can associatewith each other directly by interacting and binding to each other withspecificity. A tag and a probe can also associate with each otherindirectly such as when their complexation is mediated by a linkermolecule.

A computer program may be utilized to carry out one or more steps of anyof the methods disclosed herein. Another aspect of the presentdisclosure is a computer program product comprising a computer readablestorage medium having a computer program stored thereon which, whenloaded into a computer, performs or assists in the performance of any ofthe methods disclosed herein.

One aspect of the disclosure is a product of any of the methodsdisclosed herein, namely, an assay result, which may be evaluated at thesite of the testing or it may be shipped to another site for evaluationand communication to an interested party at a remote location, ifdesired. As used herein, “remote location” refers to a location that isphysically different than that at which the results are obtained.Accordingly, the results may be sent to a different room, a differentbuilding, a different part of city, a different city, and so forth. Thedata may be transmitted by any suitable means such as, e.g., facsimile,mail, overnight delivery, e-mail, ftp, voice mail, and the like.

“Communicating” information refers to the transmission of the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention as defined in theappended claims.

The foregoing describes the disclosure with reference to variousembodiments and examples. No particular embodiment, example, or elementof a particular embodiment or example is to be construed as a critical,required, or essential element or feature of any of the claims.

It will be appreciated that various modifications and substitutions canbe made to the disclosed embodiments without departing from the scope ofthe disclosure as set forth in the claims below. The specification,including the figures and examples, is to be regarded in an illustrativemanner, rather than a restrictive one, and all such modifications andsubstitutions are intended to be included within the scope of thedisclosure. Accordingly, the scope of the disclosure may be determinedby the appended claims and their legal equivalents, rather than by theexamples. For example, steps recited in any of the method or processclaims may be executed in any feasible order and are not limited to anorder presented in any of the embodiments, the examples, or the claims.

Example 1 Aptamer and Primer Constructs

Aptamer and biotinylated primer constructs with different 5′-terminalfunctional groups were produced and the differences are shown in FIG. 6.The aptamer contained a Cy3 fluorescent dye (Cy3 Phosphoramidite fromGlen Research(-[3-(4-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidityl]propyl]-3,3,3′,3′-tetramethylindocarbocyaninechloride)) at the 5′ terminus, and the primer contained two biotinresidues ((AB)₂), a (T)₈ linker, and a photocleavable moiety (PC Linkeravailable from Glen Research as a phosphoramidite(-(4,4′-Dimethoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite).For the method described in FIG. 6, the aptamer contained aphotoreactive crosslinking group referred to herein as ANA(4-azido-2-nitro-aniline), a photocleavable moiety (PC Linker), and aCy3 dye at the 5′ terminus, and the primer contained two biotin residuesand a (T)₈ linker.

Example 2 Affinity Binding Method (2 Catch Method)

a) Buffer

30 μL of a Cy3-aptamer mixture (2 nM each aptamer) was combined with 30μL of an (AB)₂-T8-PC-primer mixture (6 nM for each primer) in SB17T andincubated at 95° C. for 4 minutes, at 37° C. for 13 minutes. In aseparate reaction, 60 μL of a target protein mixture was prepared (2×concentration in SB17T). 55 μL of the target protein mixture wascombined with 55 μL of the aptamer/primer mixture in a 96-well plate(Omni-Tube Plate, Abgene #AB0407) and incubated at 37° C. for 15 minutesto achieve binding equilibrium. All following steps were performed atroom temperature unless otherwise noted.

b) Plasma, Serum or Whole Blood

30 μL of a Cy₃-aptamer mixture (2 nM each aptamer) was combined with 30μL of an (AB)2-T8-PC-primer mixture (6 nM for each primer) in SB17T andincubated at 95° C. for 4 minutes, at 37° C. for 13 minutes. In aseparate reaction, 30 μL of a 1× to 2.5× dilution of a complexbiological protein mixture (plasma, serum, whole blood) was prepared ina diluent containing Z-block competitor oligonucleotide(5′-(ACZZ)₇AC-3′, where Z=5-benzyl-dUTP, 4 μM) and incubated for 5minutes. The complex biological protein mixture was combined with 30 μLof a target protein mixture (4× concentration in SB17T). 55 μL of thetarget protein/biological matrix mixture was combined with 55 μL of theaptamer/primer mixture and incubated at 37° C. for 15 minutes to achievebinding equilibrium. All following steps were performed at roomtemperature unless otherwise noted.

c) Biotinylated Aptamer Capture and Free Protein Removal.

133 μL of streptavidin-agarose resin (Pierce Immobilized Streptavidin,#20353, 7.5% aqueous slurry) was washed twice with 200 μL SB17T byvacuum filtration through a Durapore membrane (MultiScreen-HV45,Millipore #MAHVN4550). 100 μL of the aptamer:protein mixture was addedto the washed resin and is mixed for 15 minutes. The resin was washedonce with 200 μL SB17T containing 10 μM biotin (Sigma-Aldrich, Inc.#B4501-1G) and once with 200 μL SB17T by vacuum filtration.

d) Protein Tagging and Aptamer Release

100 μL of SB17T containing 1.2 mM NHS-PEO4-biotin (Pierce #21329) wasadded to the washed resin and mixed for 20 minutes. The resin was washedfive times with 200 μL SB17T by vacuum filtration and once with 200 μLSB17T by centrifugation, resuspended in 75 μL SB17T containing 10 mMdextran sulfate (Mr ˜5000, Sigma-Aldrich #31404), and irradiated with aUV lamp (two Sylvania 350 Blacklight bulbs, 15W, sample 5 cm fromsource) 5 minutes with mixing. The resin was removed by centrifugationthrough the Durapore membrane, and the eluate with releasedaptamer:protein complexes was collected in a 1.1 mL 96-well plate (1.1mL Deep-Well plate, Marsh Biomedical #DW9611) containing 150 μL SB17T+10mM dextran sulfate.

e) Protein Capture and Free Aptamer Removal

50 μL of streptavidin resin (DynaBeads MyOne Streptavidin C1, Invitrogen#650-03, 10 mg/mL in SB17T) was added to a Durapore membrane. The 225 μLaptamer:protein mixture was added to the resin and mixed for 15 minutes.The resin was washed twice with 200 μL SB17T containing 10 mM dextransulfate, once with 200 μL SB17T by vacuum filtration, and once with 200μL SB17T by centrifugation.

f) Complexed Aptamer Release

The resin was resuspended in 90 μL Elution Buffer (2 mM NaOH, 0.1%TWEEN-20) and mixed for 5 minutes. During this time, aptamer is releasedfrom the protein:aptamer complex. The resin was removed bycentrifugation, and the eluate with released aptamer was collected. 80μL eluate was neutralized and buffered with 20 μL Neutralization Buffer(8 mM HCl, 0.5 mM Tris-HCl (pH 7.5), 0.1% TWEEN-20). Aptamer wasdetected as described in Example 4.

g) Results

A twelve point dilution series in buffer was created for eleven proteinanalytes (bFGF, Eotaxin-2, FGF7, FGF-16, GDNF, IL-7, IL-20,Lymphotactin, TARC, tPA, VEGF) starting with a 10 nM or 3 nM (bFGF,FGF7, tPA, Lymphotactin) concentration of each analyte and seriallydiluting to 33 fM with half-log dilutions (dilution factor of 3.1623).Two no protein controls were included to give a total of fourteensamples. The Cy3-aptamer mixture contained the eleven aptamers to thetarget proteins in the dilution series as well as twenty-eight controlaptamers whose target proteins were absent. Three replicate dilutionseries were prepared. The results are set forth in FIGS. 7-10.

FIGS. 7A to 7C show relative fluorescence units (RFU) versusconcentration plots (log-log dose response curve) for three of theeleven target proteins in buffer. Limit of Detection values (LOD) werecalculated for each protein as the protein concentration giving a signalequal to the average plus two standard deviations of the no-proteinvalues). LODs for the three proteins were 630 fM (bFGF), 90 fM (FGF7)and 530 fM (Lymphotactin). The affinity assay is able to detect proteinsin buffer at sub-picomolar levels.

FIG. 8 shows a relative fluorescence units (RFU) versus concentrationplot for three replicate measurements for the target proteinLymphotactin in buffer. The three lines represent dose response curvesfor each of the three replicates. The replicate curves are in very goodagreement with each other indicating a high level of reproducibility forthe affinity assay protocol.

A twelve point dilution series was also conducted in 10% plasma for fiveprotein analytes (bFGF, Eotaxin-2, Lymphotactin, tPA and VEGF) startingwith a 10 nM (VEGF and Eotaxin-2) or 3 nM (bFGF, tPA, Lymphotactin)concentration of each analyte and serially diluting to 420 fM or 126 fMwith 2.5-fold dilutions. Two no-protein controls were included to give atotal of fourteen samples that were subsequently hybridized on amicroarray slide. The Cy3-aptamer mixture contained the five aptamers tothe target proteins in the dilution series as well as five controlaptamers whose target proteins were absent. The assay was performed asdescribed above with the following exceptions. 100% PPT-plasma (pooledhuman plasma) was diluted 1 to 2 with 5 μM Z-block in 0.5×SB18, 0.05%TWEEN-20. 40 μL of this 50% plasma solution was mixed with 60 μL of aprotein mixture at 3.33× the final concentration. 50 μL of theplasma/protein mixture was combined with 50 μL of aptamer/primer mixture(3 nM aptamer, 9 nM primer). The equilibration binding reaction wasperformed at 37° C. for 15 min. 40 μL of the whole blood-protein-aptamermixture (instead of 100 μL) was added to the streptavidin-agarose resinand mixed for 15 min.

FIG. 9 shows a relative fluorescence units (RFU) versus concentrationplot for the target protein Lymphotactin in 10% human plasma. This curveis similar in shape and response to the dose response curves in buffer.This shows that the affinity assay protocol can be performed, but is notlimited to 10% plasma solution.

FIG. 10 shows a relative fluorescence units (RFU) versus concentrationplot for the target protein Lymphotactin in 10% whole human blood. Thiscurve is similar in shape and response to the dose response curves in10% human plasma (FIG. 9), and demonstrates the performance of theaffinity assay protocol in complex biological matrices without anyapparent matrix effects.

Example 3 Photo-Crosslink Assay Protocol

All steps of this protocol were performed with minimal light exposure toprevent photoactivation of the photoaptamer.

a) Protein Binding

30 μL of an ANA-PC-Cy3-aptamer mixture (2 nM each aptamer) was combinedwith 30 μL of an (AB)2-T8-primer mixture (6 nM for each aptamer) in SB17T Buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mMEDTA, 0.05% TWEEN-20) and incubated at 95° C. for 4 minutes, at 37° C.for 13 minutes. In a separate reaction, 60 μL of a protein mixture wasprepared at a 2× concentration. 55 μL of the target protein mixture wascombined with 55 μL of the aptamer/primer mixture in a 96-well plate(Omni-Tube Plate, Abgene #AB0407) and incubated at 37° C. for 15 minutesto achieve binding equilibrium. All following steps were performed atroom temperature unless otherwise noted.

b) Kinetic Challenge and Photo-Crosslinking

100 μL of equilibrated sample was added to 1400 μL SB17T containing 10mM dextran sulfate (Mr ˜5000, Sigma-Aldrich #31404) and incubated at 37°C. for 15 minutes. The 1.5 mL sample was irradiated with 470 nm light(Custom LED array) at 37° C. for 10 minutes to covalently crosslink thebound proteins to the photoaptamers.

c) Biotinylated Aptamer Capture and Free Protein Removal

40 μL of streptavidin resin (DynaBeads MyOne Streptavidin C1, Invitrogen#650-03, 10 mg/mL in SB17T) was added to the 1.5 mL sample and incubatedat 25° C. for 30 minutes with mixing. The resin was pelleted bycentrifugation and 1.4 mL of supernatant was removed. The resin andremaining supernatant were transferred to a Durapore membrane(MultiScreen-HV45, Millipore #MAHVN4550) and the supernatant was removedby vacuum filtration. The resin was washed twice with 200 μL SB17Tcontaining 10 μM biotin (Sigma-Aldrich, Inc. #B4501-1G) and once with200 μL SB17T by vacuum filtration.

d) Protein Tagging and Aptamer (Free and Complexed) Release

100 μL of SB17T containing 1.2 mM NHS-PEO4-biotin (Pierce #21329) wasadded to the washed resin and mixed for 20 minutes. The resin was washedthree times with 200 μL Guanidine Wash Buffer (3M guanidine, 50 mM NaCl,40 mM HEPES pH 7.5, 2 mM EDTA, 0.05% TWEEN-20, 1 mM TROLOX), and twicewith 200 μL HEPES Wash Buffer (50 mM NaCl, 40 mM HEPES pH 7.5, 0.05%TWEEN-20, 1 mM TROLOX) by vacuum filtration. The resin was resuspendedin 110 μL 20 mM NaOH and mixed for 5 minutes. The resin was removed bycentrifugation, and the NaOH eluate with released aptamer:proteincomplexes was collected. 100 μL of eluate was neutralized with 25 μL 80mM HCl, and buffered with 10 μL 55 mM HEPES (pH 7.5) containing 2M NaCland 1% TWEEN-20.

e) Protein Capture and Free Aptamer Removal

133 μL of streptavidin resin (Pierce Immobilized Streptavidin, #20347,10% aqueous slurry) was washed twice with 200 μL SB17T by vacuumfiltration through a Durapore PVDF membrane. The 135 μL aptamer:proteinmixture was added to the washed resin and mixed for 20 minutes. Theresin was washed once with 200 μL Guanidine Wash Buffer at 50° C. for 10minutes with mixing, once with 200 μL 20 mM NaOH for 2 minutes withmixing, twice with 200 μL SB17T by vacuum filtration, and once with 200μL SB17T by centrifugation.

f) Photo-Crosslinked Aptamer Release

The resin was resuspended in 100 μL SB17T and irradiated with a UV lamp(two Sylvania 350 Blacklight bulbs, 15W, sample 5 cm from source) for 20minutes with mixing. During this time, aptamer photo-crosslinked toprotein is released by photocleavage. The resin was removed bycentrifugation through the Durapore membrane, and the eluate withreleased aptamer was collected.

Example 4 Microarray Detection Protocol

a) Sample Preparation

30 μL of 4× Hybridization Buffer (3.638 M NaCl, 200 mM Na-phosphate, pH7.5, 1 nM corner marker oligo, 4 mM TROLOX, 0.1% TWEEN-20) was added to90 μL of assay sample (product of step e of the Example 2 or step f ofExample 3).

b) Microarray Slides

A ProPlate Slide Module (CSW Gasket, FLC adhesive; Grace Bio-Labs,#204841) was assembled with a microarray slide containing 14 (7×2)arrays spaced 9 mm apart. Each array consisted of three replicas of 96amine modified oligonucleotides complementary to the random region ofthe aptamers. The oligonucleotides were spotted with a contact printerin house on proprietary 3′×1′ polymer slides.

c) Microarray Blocking

100 μL of Blocking Buffer (Blocker Casein in PBS, Pierce #37528, 1 mMTROLOX) was added to the wells of the ProPlate Slide Module andincubated at 65° C. for 15-30 minutes. The Blocking Buffer was removed.

d) Hybridization and Washing

110 μL of assay sample was added to the microarray, and a 3×1×0.125 inchaluminum block was placed on top of the ProPlate Slide Module. Theassembly was wrapped in aluminum foil and incubated at 65° C. for 16hours without mixing in a humidity chamber. The Al-foil and Al-blockwere removed along with the assay sample, and the microarray was rinsedonce with 200 μL of Wash Buffer 1 (50 mM Na-phosphate, pH 7.5, 0.1%TWEEN-20), preheated to 65° C. Wash Buffer 1 was removed and theProPlate Slide Module was disassembled. The microarray slide was placedin a pap jar containing 25 mL Wash Buffer 1 (preheated to 65° C.) andincubated at 65° C. for 15 minutes with mixing. The microarray slide wastransferred to a second pap jar containing 25 mL Wash Buffer 2 (50 mMNa-phosphate, pH 7.5, preheated to 65° C.) and incubated at 65° C. for 5minutes with mixing. The microarray slide was transferred to a third papjar containing 25 mL Wash Buffer 2 and incubated at 65° C. for 5 minuteswith mixing. The microarray slide was removed from Wash Buffer 2 andimmediately dried in a stream of dry nitrogen.

e) Detection.

The microarray slide was scanned with a TECAN LS300 ReloadedFluorescence Laser Scanner, and fluorescence signal was quantified oneach feature using the software package ArrayVision (8.0 Rev 3.0,Imaging Research, Inc.). The fluorescence signal was quantified usingdensity as principal measure, with segmentation and variable spot shape.The xml export file was imported into a database for further dataanalysis.

f) Quantitative PCR Detection Protocol

Primer Design

Amplification primers for each aptamer were chosen using PrimerQuest(Integrated DNA Technologies) with default parameter settings exceptprimer Tm min=60° C., optimum=65° C., and max=70° C., and product sizerange=50-100 bp. Candidate primers were than analyzed for internalhairpin, homo-dimer, and hetero-dimer 3′ end complementarity, withOligoAnalyzer 3.0 (Integrated DNA Technologies) with default parametersettings except oligo conc.=0.2 μM. Candidates were rejected if 3′ endcomplementarity ΔG≦−3.5 kcal/mol.

Quantitative PCR Reaction

5 μL of neutralized assay sample (see step 5 of the Affinity AssayProtocol (Example 2) or step 6 of the Photo-Crosslink Assay Protocol(Example 3)) was diluted 20× with 95 μL dH2O. 20 μL amplificationreactions were prepared with 5 μL of diluted assay sample and 1×KODBuffer (Novagen #), 0.2 mM each dATP, dCTP, dGTP, and dTTP, 1×SYBR GreenI (Invitrogen #), 0.2 μM each 5′ and 3′ primer, and 0.025 U/μL KOD XLDNA Polymerase (Novagen #). Samples were prepared in a bio hood withcontaminant-free reagents. One pair of primers was used in each reactionfor quantification of one aptamer. Samples with known quantities ofaptamer were also prepared for generating standard curves. Samples wereamplified in a Bio-Rad iCyler by incubating at 95° C. for 2 minutes,cycling 40 times at 95° C. for 15 seconds followed by 72° C. for 60seconds.

Data Analysis

For each aptamer, threshold cycle (Ct) values were determined for eachsample from the amplification plots, and used to generate a standardcurve for each aptamer with the data analysis software supplied with theBio-Rad iCycler. The number of copies of each aptamer in each assaysample was determined using the standard curve, and converted to aptamerconcentration after adjusting for dilution factor and sample volume. Theconcentration of aptamer in each assay sample was plotted as a functionof input protein concentration.

Example 5 Protein Detection in Buffer Using Photo-Crosslink AssayProtocol and Array Detection

A ten point dilution series in buffer was created for thirteen proteinanalytes (angiogenin, BLC, C3a, Coagulation Factor V, Coagulation FactorXI, CTACK, Endostatin, FGF7, IGFBP-3, Prekallikrein, PSA-ACT, TIMP-1,and tPA) starting with a 10 nM concentration of each analyte andserially diluting to 330 fM with half-log dilutions (dilution factor of3.1623). Four no-protein controls were included to give a total offourteen samples. The Cy3-aptamer mixture contained the thirteenaptamers to the target proteins in the dilution series, along withfourteen control aptamers whose target proteins were absent. Sampleswere processed using the Photo-Crosslink Assay Protocol (Example 3) andquantified with Microarray Detection as described in Example 4. Theresults are set forth in FIG. 11 which shows a relative fluorescenceunits (RFU) versus concentration plot for the target protein Angiogeninin buffer.

Example 6 Protein Detection in Buffer using Affinity Assay Protocol andQ-PCR

A ten point dilution series in buffer was created for twelve proteinanalytes (angiogenin, C1q, C5b, 6 Complex, CMP-SAS, EG-VEGF, IP-10,PAI-1, PDGF-BB, Prothrombin, E-selectin, tPA, and vWF) starting with a10 nM concentration of each analyte and serially diluting to 330 fM withhalf-log dilutions (dilution factor of 3.1623). Four no-protein controlswere included to give a total of fourteen samples. The Cy3-aptamermixture contained the twelve aptamers to the target proteins in thedilution series. Samples were processed using the Affinity AssayProtocol (Example 2) and quantified by Q-PCR (Example 4). Primers2175-47-F3 (5′-GAGTGTGTGACGAGTGTGGAG-3′) (SEQ ID NO:1) and 2175-47-R3(5′-TCGGTTGTGGTGACGCCCG-3′) (SEQ ID NO:2) were used for quantificationof the angiogenin aptamer 2175-47 in the assay samples. The results areset forth in FIG. 12 which shows a log plot of the concentration ofaptamer detected versus the concentration of input protein forangiogenin.

Example 7 Protein Measurements in Test Samples are Enabled by Aptamerswith Slow Off-Rates

Preparation of Aptamer/Primer Mixtures and Test Samples

Aptamers with a biotin Cy3 detection label (4 nM each) are mixed with a3× excess of capture probe (oligonucleotide complementary to the 3′fixed region of the aptamer containing a biotin tag and photocleavableelement) in 1×SB 17T. and heated at 95° C. for 4 minutes then 37° C. for13 minutes, and diluted 1:4 in 1×SB17T. 55 uL of aptamer/primer mix isadded to a microtiter plate (Hybaid # AB-0407) and sealed with foil.Test samples are prepared in a microtiter plate by mixing knownconcentrations of protein analytes in SB 17T and diluting serially withSB17T.

Sample Equilibration

55 uL of aptamer/primer mix is added to 55 uL of test sample andincubated at 37° C. for 15 minutes in a foil-sealed microtiter plate.The final concentration of each aptamer in the equilibration mixture is0.5 nM. After equilibration, all subsequent steps of this method areperformed at room temperature unless otherwise noted.

Aptamer Capture and Free Protein Removal

A DuraPore filtration plate (Millipore HV cat# MAHVN4550) is washed oncewith 100 uL 1×SB17T by vacuum filtration, add 133.3 uL 7.5%Streptavidin-agarose resin (Pierce) is added to each well and washedtwice with 200 uL 1×SB17T. 100 uL of equilibrated samples is transferredto the Durapore plate containing the Streptavidin-agarose resin andincubated on a thermomixer (Eppendorf) at 800 rpm for 5 minutes. Theresin is washed once with 200 uL 1×SB17T+100 uM biotin and once with 200uL 1×SB17T.

Protein Tagging with Biotin

100 uL of 1.2 mM NHS-PEO4-biotin in SB17T, prepared immediately beforeuse, is added to the resin with captured aptamer and aptamer:proteincomplexes and incubated on a thermomixer at 800 rpm for 20 minutes. Theresin is washed five times with 200 uL 1×SB17T by vacuum filtration.

Slow-Off Rate Enrichment Process & Photocleavage

The drip director is removed from underside of the DuraPore plate andthe plate is placed over a 1 mL microtiter collection plate. The resinis washed once with 200 uL 1×SB17T by centrifugation at 1000×g for 30sec. 80 uL of 1×SB17T+10 mM dextran sulfate is added to the resin andirradiated with a BlackRay Mercury Lamp on a thermomixer at 800 rpm for10 minutes. The DuraPore plate is transferred to a new 1 mL deepwellplate and centrifuged at 1000×g for 30 seconds to collect thephotocleaved aptamer and protein:aptamer complexes.

Protein Capture and Free Aptamer Removal

50 uL of MyOne-streptavidin C1 paramagnetic beads (Invitrogen) (10 mg/mLin 1×SB 17T) is added to a microtiter plate. The beads are separate witha magnet for 60 seconds and the supernatant is removed. 225 uL ofphotocleavage mixture is added to the beads and mixed for 5 minutes. Thebeads are washed four times with 200 uL 1×SB 17T by separating themagnetic beads and replacing the wash buffer. The final wash buffer isremoved.

Aptamer Elution

100 uL Sodium Phosphate Elution Buffer (10 mM Na₂HPO₄, pH 11) is addedto the beads and mixed for 5 minutes. 90 uL of eluate is transferred toa microtiter plate and neutralized with 10 uL Sodium PhosphateNeutralization Buffer (10 mM NaH₂PO₄, pH 5).

Aptamer Hybridization to Microarrays

DNA arrays are prepared with oligonucleotide capture probes comprised ofthe complementary sequence of the variable region of each aptamerimmobilized on a custom microscope slide support. Multiple arrays(subarrays) exist on each slide, and subarrays are physically separatedby affixing a gasket (Grace) for sample application. Arrays arepretreated with 100 uL Blocking Buffer and incubated for 15 minutes at65° C. on a thermomixer. 30 uL of Hybridization Buffer is added to 90 uLof neutralized aptamer eluate in a microtiter plate, incubated at 95° C.for 5 minutes in a Thermal Cycler, and cooled to 65° C. at 0.1°C./second. Blocking Buffer is removed from the arrays and 110 uL ofaptamer sample is added to the arrays and incubate in a humid chamber at65° C. for 20 hours.

Array Washing

Aptamer sample is removed from the arrays and the arrays are washed oncewith 200 uL of sodium phosphate Tween-20 wash buffer at 65° C., with thegasket in place, and three times with 25 mL sodium phosphate, Tween-20wash buffer at 65° C. in a pap jar with the gasket removed. Arrays aredried with a nitrogen gun.

Quantitate Signal on Arrays

Array slides are scanned on a TECAN LS300 Reloaded.in an appropriatechannel for Cy3 detection and Cy3 signal on each array feature isquantified.

Results:

Apatmers specific to three different targets (bFGF, VEGF, andMyeloperoxidase) were produced using traditional SELEX methods andmaterials. A second set of aptamers specific to the same set of targetswere made using 5-position modified nucleotides and selected for veryslow off-rates for their respective targets. Aptamers made in thetraditional process had measured off rates on the order of less than 5minutes. Aptamers made with the modified nucleotides and using slowoff-rate enrichment process during selection had off rates of greaterthan 20 minutes. Two sets of aptamers were made for each target by thetwo different methods for a total of 4 different aptamer populations foreach target. The ability of these aptamer populations to measure analyteconcentrations in test samples was evaluated as described above over arange of target concentrations. Relative signal from the DNA chipdetection was plotted against the input target concentration. See FIG.13A to 13C. The response curve of the traditional aptamers is very flatand the sensitivity of the detection is fairly low. The sensitivity ofdetection of the respective targets with the slow off-rate aptamers isexcellent. The data supports the need to use the slow-off aptamers formaximum analytic performance.

Example 8 Reproducibility Using Slow Off Rate Aptamers

Using the method of Example 7, one plasma sample was split into 68different aliquots. The assay of Example 7 was performed on each of the68 samples. 34 of the samples were combined and split again. Theremaining 34 samples were simply retested. In this manner thereproducibility of the assay can be tested for within and between assayconsistency. The sample layout and detection scheme is shown in FIG. 14.FIG. 15 shows the % CV in the measurement of all the samples indicatedin FIG. 14. The unpooled and pooled samples have the same CV's.

Example 9 One Catch Affinity Binding Method

a) Equilibration of Aptamers with Plasma, Serum, or Whole Blood

30 uL of a Cy3-aptamer mixture (20 nM each aptamer) is combined with 30uL of an (AB)₂-T₈-PC-primer mixture (60 nM for each primer) in SB17T andincubated at 95° C. for 4 minutes and at 37° C. for 13 minutes. 55 uL ofthe complex biological protein mixture (plasma, serum, or whole blood),diluted 1:1000 in SB17T, is combined with 55 uL of the aptamer/primermixture and incubated at 37° C. for 15 minutes to achieve bindingequilibrium. All following steps are performed at room temperatureunless otherwise noted.

b) Protein Tagging

100 uL of aptamer:protein mixture is combined with 10 uL of SB17Tcontaining 500 uM NHS-PEO4-biotin (Pierce #21329) and incubated for 20minutes at 37° C. Excess NHS reagent is quenched by adding 10 uL of 200mM TRIS buffer (pH 7.5) to the reaction mixture and incubating for 10minutes at 37° C.

c) Protein Capture and Free Aptamer Removal

100 uL of streptavidin resin (DynaBeads MyOne Streptavidin C1,Invitrogen #650-03, 10 mg/mL in SB 17T) is added to a Durapore membraneto capture aptamer/protein complexes. 100 uL of the aptamer/proteinmixture is added to the resin, mixed for 15 minutes, and vacuum filteredto remove free aptamer. The resin is washed three times with 200 uL SB17T by vacuum filtration, and once with 200 uL SB 17T by centrifugation.

d) Complexed Aptamer Release

The resin is resuspended in 90 uL Elution Buffer (2 mM NaOH, 0.1%TWEEN-20) and mixed for 5 minutes to release aptamer from theaptamer/protein complex. The resin is removed by centrifugation, and theeluate containing released aptamer is collected. 80 uL eluate isneutralized and buffered with 20 uL Neutralization Buffer (8 mM HCl, 0.5mM Tris-HCl (pH 7.5), 0.1% TWEEN-20). Aptamer is detected as describedin Example 4.

A number of patents, patent application publications, and scientificpublications are cited throughout and/or listed at the end of thedescription. Each of these is incorporated herein by reference in theirentirety. Likewise, all publications mentioned in an incorporatedpublication are incorporated by reference in their entirety.

Examples in cited publications and limitations related therewith areintended to be illustrative and not exclusive. Other limitations of thecited publications will become apparent to those of skill in the artupon a reading of the specification and a study of the drawings.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively.

1-76. (canceled)
 77. A method for detecting for the presence of a targetmolecule in a test sample, the method comprising: (a) preparing amixture by contacting the test sample with an aptamer comprising a firsttag and having specific affinity for the target molecule, wherein anaptamer affinity complex is formed if the target molecule is present inthe test sample; (b) exposing the mixture to a first solid supportcomprising a first capture element, and allowing the first tag toassociate with the first capture element; (c) removing components of themixture not associated with the first solid support; (d) releasing theaptamer affinity complex from the first solid support; (e) attaching asecond tag to the target molecule of the aptamer affinity complex; (f)exposing the released aptamer affinity complex to a second solid supportcomprising a second capture element and allowing the second tag toassociate with the second capture element; (g) removing uncomplexedaptamer from the mixture by partitioning the uncomplexed aptamer fromthe aptamer affinity complex; (h) detecting for the presence of thetarget molecule by detecting the aptamer portion of the aptamer affinitycomplex; and wherein, the method is capable of detecting for thepresence of the target molecule in the test sample when the targetmolecule is present at a sub-picomolar level.
 78. The method of claim77, wherein the aptamer comprises at least one modified nucleotide. 79.The method of claim 78, wherein the at least one modified nucleotide isa C-5 modified pyrimidine.
 80. The method of claim 79, wherein the C-5modified pyrimidine is selected from the group listed in FIG.
 16. 81.The method of claim 79, wherein the aptamer further comprises at leastone chemical modification at one or more positions independentlyselected from the group consisting of a 2′-position sugar modification,a 2′-amino (2′—NH₂), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a5-position pyrimidine modification, an 8-position purine modification, amodification at a cytosine exocyclic amine, a substitution of5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap,and a 5′ cap.
 82. The method of claim 77, wherein (h) further comprisesdissociating the aptamer from said aptamer affinity complex beforedetecting the aptamer.
 83. The method of claim 77 further comprisingdiluting the mixture containing the aptamer affinity complex at anypoint after (a) and before (d) and incubating the mixture containing theaptamer affinity complex for a time selected from the group consistingof greater than or equal to 30 seconds, 1 minute, 2 minutes, 3 minutes,4 minutes, 5 minutes, 10 minutes, 30 minutes, and 60 minutes.
 84. Themethod of claim 77 further comprising adding a competitor to the mixturecontaining the aptamer affinity complex at any point after (a) andbefore (d) and incubating the mixture containing the aptamer affinitycomplex for a time selected from the group consisting of greater than orequal to 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 10 minutes, 30 minutes, and 60 minutes.
 85. The method of claim84, wherein the competitor is selected from the group consisting of anoligonucleotide, heparin, herring sperm DNA, salmon sperm DNA, dextransulfate, polyanion, abasic phosphodiester polymer, dNTP, andpyrophosphate.
 86. The method of claim 77, wherein the rate ofdissociation of the aptamer affinity complex (t_(1/2)) is selected fromthe group consisting of ≧30 minutes, ≧60 minutes, ≧90 minutes, ≧120minutes, ≧150 minutes, ≧180 minutes, ≧210 minutes, and ≧240 minutes. 87.The method of claim 77, wherein the aptamer is detected and optionallyquantified using a method selected from the group consisting of Q-PCR,MS, and hybridization.
 88. The method of claim 77 further comprisingadding a detectable moiety to the aptamer.
 89. The method of claim 88,wherein the detectable moiety is selected from the group consisting of adye, a quantum dot, a radiolabel, a electrochemical functional group, anenzyme, and an enzyme substrate.
 90. The method of claim 77, wherein theaptamer comprises DNA, RNA or both DNA and RNA.
 91. The method of claim77, wherein the target molecule is selected from the group consisting ofa protein, a peptide, a carbohydrate, a polysaccharide, a glycoprotein,a hormone, a receptor, an antigen, an antibody, a virus, a substrate, ametabolite, a transition state analog, a cofactor, an inhibitor, a drug,a dye, a nutrient, a growth factor, a tissue, and a controlledsubstance.
 92. The method of claim 77, wherein the test sample isselected from the group consisting of a biological sample, anenvironmental sample, a chemical sample, a pharmaceutical sample, a foodsample, an agricultural sample, and a veterinary sample.
 93. The methodof claim 92, wherein the biological sample is selected from the groupconsisting of whole blood, leukocytes, peripheral blood mononuclearcells, plasma, serum, sputum, breath, urine, semen, saliva, meningialfluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate,bronchial aspirate, synovial fluid, joint aspirate, cells, a cellularextract, stool, tissue, a tissue extract, a tissue biopsy, andcerebrospinal fluid.
 94. The method of claim 77, wherein the first tagand the second tag each comprise at least one component independentlyselected from the group consisting of a polynucleotide, a polypeptide, apeptide nucleic acid, a locked nucleic acid, an oligosaccharide, apolysaccharide, an antibody, an affybody, an antibody mimic, a cellreceptor, a ligand, a lipid, biotin, avidin, streptavidin, Extravidin,neutravidin, a metal, histidine, and any portion of any of thesestructures.
 95. The method of claim 77, wherein the first captureelement and said second capture element each comprises at least onecomponent independently selected from a polynucleotide, a polypeptide, apeptide nucleic acid, a locked nucleic acid, an oligosaccharide, apolysaccharide, an antibody, an affybody, an antibody mimic, a cellreceptor, a ligand, a lipid, biotin, avidin, streptavidin, Extravidin,neutravidin, a metal, histidine, and any portion of any of thesestructures.
 96. The method of claim 77, wherein the first tag comprisesa releasable moiety.
 97. The method of claim 77, wherein the first solidsupport and the second solid support each is independently selected fromthe group consisting of a polymer bead, an agarose bead, a polystyrenebead, an acrylamide bead, a solid core bead, a porous bead, aparamagnetic bead, glass bead, controlled pore bead, a microtitre well,a cyclo-olefin copolymer substrate, a membrane, a plastic substrate,nylon, a Langmuir-Blodgett film, glass, a germanium substrate, a siliconsubstrate, a silicon wafer chip, a flow through chip, a microbead, apolytetrafluoroethylene substrate, a polystyrene substrate, a galliumarsenide substrate, a gold substrate, and a silver substrate.
 98. Themethod of claim 77, wherein the method has a limit of detection (LOD)selected from the group consisting of 630 fM, 530 fM and 90 fM.
 99. Themethod of claim 77, wherein the method is capable of detecting thetarget molecule FGF7 in a test sample with an LOD of 90 fM.
 100. Themethod of claim 77, wherein the method is capable of detecting thetarget molecule Lymphotactin in a test sample with an LOD of 530 fM.101. The method of claim 77, wherein the method is capable of detectingthe target molecule bFGF a test sample with an LOD of 630 fM.
 102. Themethod of claim 77, wherein said kinetic challenge comprises dilutingthe mixture containing the aptamer affinity complex at any point after(a) and before (d) and incubating the mixture containing the aptameraffinity complex for a time such that the ratio of the measured level ofaptamer affinity complex to the measured level of the non-specificcomplex is increased.
 103. The method of claim 77, wherein said kineticchallenge comprises adding a competitor to the mixture containing theaptamer affinity complex at any point after (a) and before (d) andincubating the mixture containing the aptamer affinity complex for atime such that the ratio of the measured level of aptamer affinitycomplex to the measured level of the non-specific complex is increased.